Membrane scaffold proteins and tethered membrane proteins

ABSTRACT

Membrane proteins are difficult to express in recombinant form, purify, and characterize, at least in part due to their hydrophobic or partially hydrophobic properties. Membrane scaffold proteins (MSP) assemble with target membrane or other hydrophobic or partially hydrophobic proteins or membrane fragments to form soluble nanoscale particles which preserve their native structure and function; they are improved over liposomes and detergent micelles. In the presence of phospholipid, MSPs form nanoscopic phospholipid bilayer disks, with the MSP stabilizing the particle at the perimeter of the bilayer domain. The particle bilayer structure allows manipulation of incorporated proteins in solution or on solid supports, including for use with such surface-sensitive techniques as scanning probe microscopy or surface plasmon resonance. The nanoscale particles facilitate pharmaceutical and biological research, structure/function correlation, structure determination, bioseparation, and drug discovery.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.09/990,087, filed Nov. 20, 2001 (now U.S. Pat. No. 7,048,949, issued May23, 2006), which claims benefit of U.S. Provisional Application No.60/252,233, filed Nov. 20, 2000.

ACKNOWLEDGMENT OF FEDERAL RESEARCH SUPPORT

This invention was made, at least in part, with funding from theNational Institutes of Health (Grant Nos. (R01GM31756, R01GM33775,GM63574 and 5F32GM19024). Accordingly, the U.S. Government has certainrights in this invention.

BACKGROUND OF THE INVENTION

The field of present invention encompasses molecular biology andmembrane technology. Specifically, the present invention relates toartificial membrane scaffold proteins (MSPs), sequences encoding them,vectors and recombinant host cells, methods for recombinant productionof them, and methods of using the membrane scaffold proteins tostabilize, disperse and solubilize fully or partially hydrophobicproteins such as tethered, embedded or integral membrane proteins whilemaintaining the biological activities of those membrane proteins or tostabilize, disperse and solubilize membrane fragments.

Several years ago we developed a new system for the study of membraneproteins by scanning probe microscopy, based on the adsorption ofsynthetic high density lipoprotein disks (rHDL, apo A-I) onto mica in anoriented manner (Carlson et al., 1997; Bayburt et al., 1998; Bayburt etal., 2000; Carlson et al., 2000). The diameters of the discoidalstructures observed are approximately 10 nm with a height of 5.5nanometers. The 5.5 nm high topology observed is most compatible with asingle membrane bilayer epitaxially oriented on the atomically flat micasurface (Carlson et al., 1997).

Purified membrane proteins can be reconstituted into the phospholipidbilayer domain of certain such discoidal structures and studied insolution or subsequently adsorbed on a suitable surface for eitheratomic force microscopy or examination by spectroscopic techniques thattake advantage of a surface of oriented protein-bilayer assemblies.Additionally, the underlying discoidal structures containing themembrane protein are easily recognizable and provide a point ofreference for judging the quality of the sample and images. A tetheredmembrane protein, NADPH-cytochrome P450 reductase, was incorporated andphysically studied in rHDL bilayer disks (Bayburt et al., 1998; Bayburtet al., 2000). The reductase can be incorporated into 10 nm diameterrHDL disks, those disks can be absorbed onto mica, and the catalyticdomain of the reductase, which protrudes from the top of the bilayerstructure, can be imaged. The incorporated enzyme is active on such asurface, with a turnover number consistent with that obtained withparticulate membrane preparations. Force curve analysis has been used toestimate the height of the domain and its compressibility under theforce of the AFM probe (Bayburt et al., 2000). The height of themolecule above the bilayer surface corresponds to the predicted heightbased on the recent X-ray crystal structure (Wang et al., 1997).Cytochrome P450 reductase can be incorporated in active form inMSP-supported nanoscale structures of the present invention.

High-density lipoproteins (HDL) are assemblies of a protein component,termed apo A-I, and various phospholipids. HDL particles play animportant role in mammalian cholesterol homeostasis by acting as theprimary conduit for reverse cholesterol transport (Fielding andFielding, 1991). The function of HDL as a cholesterol transporter reliesupon the activity of the HDL-associated enzyme lecithin-cholesterol acyltransferase, or LCAT (Glomset, 1968; Jonas, 1991), which mediates theinsertion of cholesterol esters into HDL lipoprotein particles. Certainportions of the apo A-I protein are required for the activity of thisenzyme (Holvoet et al., 1995). In addition, a part of the apo A-Iprotein is thought to be in a globular domain at the N-terminus, and tobe responsible for interactions with cell surface receptors. One nascentform of HDL particles has been assumed to be that of a discoid based onelectron microscopy of stained preparations (Forte et al., 1971). Ourlaboratory has confirmed this using AFM studies of synthetic forms ofrHDL under aqueous conditions. This form, however, is not thepredominant form in circulation in vivo. Rather, the apo A-I structureappears to have evolved to stabilize a spherical form.

Two general models for the structure of HDL disks have been proposed.One model has the apo A-I protein surrounding a circular bilayer sectionas a horizontal band or “belt” composed of a curving segmented alphahelical rod (Wlodawer et al., 1979). The other model has the proteintraversing the edges of the bilayer vertically in a series of alphahelical segments (Boguski et al., 1986). Both models are based primarilyon indirect experimental evidence, and no definitive means ofdistinguishing between them has emerged. Sequence analysis of the apoA-I genes suggests that the protein folds into a series of helicesroughly 22 amino acids long, which is consistent with roughly a bilayerthickness. The placement of the helices in the disks has been predictedby computer modeling (Phillips et al., 1997) and attenuated totalreflectance infrared spectroscopic measurements (Wald et al., 1990).These efforts suggested the helices lie roughly parallel to the acylchains and are slightly shorter than the thickness of a bilayer. Thisarrangement of proteins and lipid is consistent with the picket fencemodel.

A belt model is consistent with some electron microscopy and neutronscattering data (Wlodawer et al., 1979), where the helices are arrangedlongitudinally around the edge of the bilayer disks like a “belt”. Morerecent infrared spectroscopy studies using a new method of sampleorientation for dichroism measurements are more consistent with the beltmodel, in contrast to earlier studies (Wald et al., 1990; Koppaka etal., 1999). So far, there is no compelling direct evidence as to whichmodel is correct, even though a low resolution x-ray crystal structurefor apo A-I crystallized without lipid (Borhani et al., 1997) has beenobtained. The low resolution crystal structure of an N-terminallytruncated apo A-I shows a unit cell containing a tetrameric speciescomposed of 4 helical rods which bend into a horseshoe shape and whichcombine to give a circular aggregate about 125×80×40 Å. It was suggestedthat a dimeric species in this belt conformation is capable of formingdiscoidal particles.

The information collected to date concerning the reverse cholesteroltransport cycle and the maturation of HDL particles suggests that theapo A-I protein has unique properties that allow it to interactspontaneously with membranes resulting in the formation of lipoproteinparticles. Initial apo A-I lipid binding events have been proposed(Rogers et al., 1998), but conversion of bilayer-associated forms todiscoidal particles remains unclear. The available evidence suggeststhat the energy of stabilization of lipid-free apo A-I is fairly low andthat there is an equilibrium between two conformers (Atkinson and Small,1986; Rogers et al., 1998). One conformer may be a long helical rod, andthe other may be a helical “hairpin” structure about half as long. Ithas been suggested that the low stabilization energy and conformationalplasticity allow apo A-I to structurally reorganize when it encounters alipid membrane, thus facilitating the structural changes that would haveto take place in both the membrane and the protein to produce discreetlipoprotein particles (Rogers et al., 1998). Once these particles areformed, apo A-I may still undergo specific conformational changes thatcontribute to the dynamic functionality of the lipoprotein particles.All of these interactions and changes take place at the protein-lipidinterface. Thus, there is little reason to believe that apo A-I itselfwould be ideal for generating a stable, nanometer size phospholipidbilayer.

Synthetic rHDL form spontaneously upon interaction of apo A-I withphosphatidylcholine liposomes at certain protein-lipid ratios andtemperatures at or above the phase-transition temperature of the lipid(Jonas, 1986). The method of detergent dialysis of mixtures of apo A-Iand phospholipid is also used to form particulate structures and affordsa method of incorporating purified membrane proteins. The sizes ofdiscoidal particles formed depend on the protein to lipid ratio of theformation mixture and reflect the diameter of the bilayer domain(Brouillette et al., 1984; Wald et al., 1990). Size classes thereforearise from the number of associated apo A-I molecules at the perimeterof the phospholipid disk. These classes have been termed LP1, LP2, LP3,and LP4 for the stoichiometry of apo A-I protein molecules per disk.Variable sizes within the LP classes also arise due to heterogeneity inthe conformation of apo A-I. One aspect of the present invention isbased on the ability to identify the structure responsible for thisheterogeneity and eliminate it to produce a monodisperse population ofdisk structures. Currently, the formation of homogeneous particleslarger than 10 nm diameter requires separation of the particles from amixture of species containing from 2 to 4 associated apo A-I molecules,while 10 nm diameter particles are the major form at low apo A-I tophospholipid ratios during formation. The purity of single size classesand the ability to obtain high efficiencies of membrane protein ormembrane fragment incorporation requires alteration of the apo A-Istructure.

Different types of lipid aggregates are used for reconstitution andstudy of purified membrane proteins; these include membrane dispersions,detergent micelles and liposomes. See FIG. 1. Purified systems forbiochemical and physical study require stability, which is not alwaysinherent in some systems. Liposomes are closed spherical bilayer shellscontaining an aqueous interior. Reconstitution into liposomes bydetergent dialysis or other methods typically results in randomorientation of the protein with respect to outer and lumenal spaces.Since ligands or protein targets are usually hydrophilic or charged,they cannot pass through the liposomal bilayer as depicted in FIG. 1,although this may be advantageous in some instances. Since both sides ofthe liposomal bilayer are not accessible to bulk solvent, couplingeffects between domains on opposite sides of the bilayer cannot bestudied. Liposomes are also prone to aggregation and fusion and areusually unstable for periods of more than about a week or under certainphysical manipulations, such as stopped flow or vigorous mixing. Thesize of liposomes obtained by extruding through defined cylindrical poresizes polydisperse in size distribution rather than exhibiting a uniformdiameter.

Liposomes also may present difficulties due to light scattering, andaggregation of membrane proteins present in the bilayer. The surfacearea of a liposome is relatively large (10⁵ to 10⁸ Å2). To obtainliposomes with single membrane proteins requires a large lipid toprotein molar ratio. Detergent micelles allow solubilization of membraneproteins by interaction with the membrane-embedded portion of theprotein and are easy to use. Detergent micelles are dynamic and undergostructural fluctuations that promoter subunit dissociation and oftenpresent difficulty in the ability to handle proteins in dilutesolutions. An excess of detergent micelles, however, is necessary tomaintain the protein in a non-aggregated and soluble state. Detergentscan also be mildly denaturing and often do not maintain the propertiesfound in a phospholipid bilayer system. Specific phospholipid speciesare often necessary to support and modulate protein structure andfunction (Tocanne et al., 1994). Thus, the structure, function, andstability of detergent solubilized membrane proteins may be called intoquestion. Since an excess of detergent micelles is needed, proteincomplexes can dissociate depending on protein concentration and thedetergent protein ratio. By contrast, the MSP nanostructures of thepresent invention are more robust structurally, having a phospholipidbilayer mimetic domain of discrete size and composition and greaterstability and smaller surface area than unilamellar liposomes. The diskstructures allow access to both sides of the bilayer like detergents,and also provide a bilayer structure like that of liposomes.

There is a long felt need in the art for stable, defined compositionsfor the dispersion of membrane proteins and other hydrophobic orpartially hydrophobic proteins, such that the native activities andproperties of those proteins are preserved.

SUMMARY OF THE INVENTION

Membrane Scaffold Proteins (MSPs) as used herein are artificial proteinswhich self assemble with phospholipids and phospholipid mixtures, or inthe absence of phospholipids, into nanometer size membrane bilayers. Asubset of these nanometer size assemblies are discoidal in shape, andare referred to as nanodisc or nanodisc structures. These “nanoscale”particles can be from about 5 to about 500 nm, about 5 to about 100 nmor about 5 to about 50 nm in diameter. These structures comprisingphospholipid and MSP preserve the overall bilayer structure of normalmembranes but provide a system which is both soluble in solution andwhich can be assembled or affixed to a variety of surfaces. The aminoacid sequences of specifically exemplified MSPs are given in SEQ IDNO:6, SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:23, SEQ IDNO:29, SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:45.

The present invention further provides the use of the nanometer scalephospholipid bilayer structures or nanodiscs formed using the MSPs ofthe present invention are useful for the incorporation of additionalhydrophobic or partially hydrophobic protein molecules. Those additionalproteins can be solubilized, for example, with the use of detergent, andthe solubilized proteins can be added to a solution of MSP, with orwithout phospholipid(s), and the nanoscale particles self assemble sothat the MSPs and the additional proteins are incorporated into a stableand soluble particle. Subsequently, any detergent can be removed bydialysis. Those proteins, found in nature in the various membranestructures of a living organism, are solubilized in the MSP supportednanobilayer or nanodisc, and the native structure and activity of thetarget protein are preserved in these MSP-supported structures. Besideshydrophobic or partially hydrophobic proteins, membrane fragments ordisrupted membranes can be assembled with the MSPs of the presentinvention.

The MSP supported bilayers or nanodiscs can be used in solutions orapplied to a number of surfaces, such that the native structure andligand binding of the natural protein incorporated in the MSP supportedstructure are maintained. As specifically exemplified, the MSP supportedstructures are affixed to a gold surface, e.g., for use in surfaceplasmon resonance technologies.

The present invention relates to methods for the incorporation ofintegral membrane proteins into nanoscale lipid bilayers or nanodiscscomprising at least one MSP of the present invention. Three classes ofmembrane proteins (tethered, embedded, and integral) can be used in themethods of the present invention. The first membrane protein class isthe tethered; this class is exemplified by NADPH-cytochrome P450reductase and human tissue factor. Examples of embedded membraneproteins include, without limitation, cytochrome P450 2B4 from rabbitliver microsomes, cytochrome P450 3A4 from human liver microsomes andcytochrome P450 6B1 from insect microsomes. The integral membraneproteins are exemplified by the 7-helix transmembrane proteins,including, but not limited to, bacteriorhodopsin from Halobacteriumhalobium, the 5-hydroxy tryptamine 1A G-protein coupled receptor fromHomo sapiens and other G-protein coupled protein receptors. Members ofeach class of membrane protein have been successfully incorporated intothe nanoscale structures using the MSPs and methods of the presentinvention. In particular, cell surface receptors, and especiallyG-protein coupled receptors, can be incorporated into nanobilayerbilayer structures formed from a class of membrane scaffold proteins(MSPs).

We developed nanodiscs for use in structural, biochemical andpharmaceutical techniques by engineering the scaffold protein (MSP) forgreater stability, size homogeneity and useful functionalities in theresultant nanoscale lipoprotein particle. These particles can form tagsfor purification and physical manipulation of disks such as in hydrogelsor on a gold biosensor surface, and they can serve as entities for rapidand reproducible assay and crystallization. The nanoparticles andmembrane protein scaffolds are useful in biotechnology, thepharmaceutical industry as well as in basic research. In addition, thestructural and functional principles uncovered through our discovery andthe related techniques facilitate understanding the interactions ofproteins with lipid bilayers at the molecular level.

Another aspect of this invention relates to a class of membrane scaffoldprotein (MSP) that can be used to solubilize membrane proteins andcomplexes in a functionally stable monodisperse phospholipid-bilayerassociated form. Furthermore, this MSP provides means of physicalmanipulation of single membrane proteins. The MSP is modeled after humanapolipoprotein A-I, which under certain conditions, can self-assemblewith phospholipid to form discoidal structures having diameters of 100to 200 Å (Brouillette et al., 1984). Other amphipathic proteins couldalso have served as a starting point. Although apolipoprotein A-I wasknown, it has not been used in any general method to solubilize andstudy tethered, embedded or integral membrane proteins of completelyunrelated origin. A specific embodiment of this invention is the use oflipid-associated MSPs for the solubilization, manipulation and study ofmembrane proteins.

The present invention further provides materials and methods usinggenetically engineered MSPs to minimize the size of the MSP with respectto membrane particle formation and to increase the stability andmonodispersity of the self-assembled nanoparticle by altering thesequence of the parent molecule. In particular, we have developed MSPsfor the study of G-protein coupled receptors. G-protein coupledreceptors (GPCRs) are an important and diverse class of pharmaceuticaltargets in mammalian cellular membranes where they function as signaltransducing elements, bind several classes of bioactive ligands andtransmit information to the intracellular machinery. The artificial MSPof the present invention stabilizes and solubilizes themembrane-associated form of GPCR to allow purification and manipulationin solution or on a solid support for use in high throughput screeningapplications and on surfaces for surface-plasmon biosensor andscanning-probe techniques. The artificial MSP of the present inventioncan be used to facilitate expression and purification of recombinantmembrane proteins in soluble form.

Also within the scope of the present invention are recombinant DNAmolecules which encode MSPs and host cells containing those recombinantDNA molecules which are used to produce the MSPs. MSPs encoded by theserecombinant DNA molecules include those comprising amino acid sequencesincluding, but not limited to, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:17,SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:43, SEQ ID NO:44 andSEQ ID NO:45.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates different types of lipid aggregatesincorporating a membrane protein. Small circles and triangles representligand for intracellular and extracellular domains of the receptorproteins, respectively.

FIG. 2 shows the wheel structure of an alpha helix, with the placementof hydrophobic and hydrophilic amino acid side chains that give thehelix its amphipathic character.

FIG. 3A is a schematic representation of a “picket fence” model for MSPstabilization of a bilayer. The circles represent single a helices witha diameter of about 1.5 nm and 0.54 nm per turn of the helix (3.6 aminoacid residues per turn). FIG. 3B is a schematic of a “belt” model of anMSP supported bilayer. The rectangles represent single a helices with adiameter of about 1.5 nm and a helix length of about 3.9 nm.

FIG. 4 presents the sequence and secondary structure prediction for theparent apo A-I molecule. Stars denote helical repeat boundaries.Underlined sequence in bold represents a-helical structure, italicsrepresents b structure and plain text represents potential turns.

FIGS. 5A-5G illustrate various engineered MSP structures, shown withpicket fence topology and helical assignments based on sequenceanalysis. FIG. 5A: MSP1 showing positions of half-repeats. Half-repeat 1is disordered based on molecular dynamics simulation (Phillips, 1997).FIG. 5B: Hinge domain movement. FIG. 5C: Removal of half-repeats. FIG.5D: Hinge domain replacement with helices 3 and 4. FIG. 5E: MSP2, with atandem duplication of the sequence of MSP1. FIG. 5F: Removal ofhalf-repeat 1 to make MSP1Δ1. FIG. 5G: Tandem repeat of MSP1Δ1 to formMSP2Δ1.

FIGS. 6A-6B diagrammatically illustrate the PCR strategy used to amplifyartificial MSPs.

FIGS. 7A-7B shows diagrams of MSP2 with (FIG. 7A) and without a longlinker sequence (FIG. 7B).

FIGS. 8A-8B illustrate the strategy for constructing and expressing anartificial sequence encoding an MSP1 derivative lacking helices 4 and 5.

FIGS. 9A-9B illustrate the strategy for constructing and expressing anartificial sequence encoding an MSP1 derivative lacking helices 5 and 6.

FIG. 10 provides a gel filtration elution profile of bacteriorhodopsinreconstituted into MSP1 structures.

FIG. 11 show a gel filtration chromatogram of bacteriorhodopsinsolubilized by MSP1 in the absence of added phospholipid.Bacteriorhodopsin is detected by absorbance at 550 nm, while MSP1 andbacteriorhodopsin protein is detected by absorbance at 280 nm.

FIG. 12 is a gel filtration chromatogram of bacteriorhodopsinsolubilized by MSP2 in the absence of added phospholipid.Bacteriorhodopsin is detected by absorbance at 550 nm, while MSP2 andbacteriorhodopsin protein is detected by absorbance at 280 nm.

FIG. 13 illustrates nanoscale particle formation with varying lipid toMSP ratios. Particles were formed at the indicated lipid to MSP moleratio and separated by native gradient gel electrophoresis. As indicatedat the right, MSP1 forms 8.2, 9.6 and 10.6 nm diameter particles. MSP2forms predominantly 9.6 nm particles.

FIG. 14 shows the results of chemical denaturation of MSP1 and MSP2particles (9 nm diameter) in complexes with dipalmitoylphosphatidylcholine as monitored by tryptophan fluorescence. Excitationwas at 280 nm, and the buffer was 10 mM Hepes pH 7.5, 0.15 M NaCl.

FIGS. 15A-15B show the membrane proteins incorporated into disks andattached to solid supports. FIG. 15A: Disk-associated receptor andligand-induced assembly of receptor-target complex on gold. FIG. 15B:Disk-associated receptor in gel matrix.

FIG. 16A shows the results of HPLC over a sizing column of nanodiscparticles with retention times indicating 8 and 10 nm sizes and TissueFactor activity at 25-28 min. FIG. 16B shows the results of SDS-PAGEusing 8-25% gradient gels; the MSP1-supported nanodisc bilayers isolatedfrom the HPLC and containing TF have the expected molecular weights.

FIGS. 17A-17B illustrate thee separation of cytochrome b5 into discs asdetermined by native PAGE using 8-25% gradient gels. Lane 1, molecularweight markers; lane 2, cytochrome b5; lane 3, disks; lane 4,disk/cytochrome b5; lanes 5 and 6, anion exchange purified cytochrome b5in disks representing two pools eluted from the anion exchange column.FIG. 17A shows protein stained with Coomassie blue, and FIG. 17B showsheme-specific staining. Note that the 232 kDa marker protein (catalase)also stained for heme.

FIG. 18 is a chromatogram of cytochrome P450 3A4 incorporated into 10 nmbilayer discs consisting of 85% DPPC, 15% POPC.

FIG. 19 is a chromatogram of cytochrome P450 3A4 incorporated into 10 nmbilayer discs consisting of 85% DPPC, 15% POPS.

FIG. 20 is a chromatogram of cytochrome P450 3A4 incorporated into 10 nmbilayer discs consisting of 100% DPPC.

FIG. 21 shows the results of PAGE over an 8-25% gradient gel with threedisc samples which correlate with the sizes of the nanodisc particles.

FIG. 22 illustrates the results of HPLC chromatography ofMSP-solubilized cytochrome P450 6B1 over a Superdex 200 sizing column.Retention times indicate nanodisc particles containing 6B1 (420 nmtrace). The flow rate was 0.25 ml/min.

FIG. 24 illustrates the results of PAGE with sample 1 (nanodiscsprepared with microsomal membranes from cells coexpressing cytochromeP450 6B1 and NADPH P450 reductase. Sample 2 contains control microsomes.

FIG. 24 provides a [CO—Fe⁺²—CO—Fe³⁺] optical difference spectrum. Activecytochrome P450 6B1 incorporated into nanodiscs absorbs at 450 nm.

FIG. 25 depicts a chromatogram of sample separated by a Superdex sizingcolumn. Retention times indicated rHDL particles 10 nm in size.

FIG. 26 illustrates co-incorporation of cytochrome P450 reductase andcytochrome P450 6B1 in MSP nanodiscs. The ratio of absorbances at 456 nm(predominantly reductase) to that at 420 nm (predominantly P450) isplotted as a function of retention time. The peak at about 26 minindicates a nanodisc population containing both reductase andcytochrome.

FIG. 27 illustrates the binding of DPPC nanodiscs containing carboxylterminated thiols to a gold surface, as monitored by surface plasmonresonance.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations used in this application include A, Ala, Alanine; M, Met,Methionine; C, Cys, Cysteine; N, Asn, Asparagine; D, Asp, Aspartic Acid;P, Pro, Proline; E, Glu, Glutamic Acid; Q, Gln, Glutamine; F, Phe,Phenylalanine; R, Arg, Arginine; G, Gly, Glycine; S, Ser, Serine; H,His, Histidine; T, Thr, Threonine; I, Ile, Isoleucine; V, Val, Valine;K, Lys, Lysine; W, Try, Tryptophan; L, Leu, Leucine; Y, Tyr, Tyrosine;MSP, membrane scaffold protein; DPPC, dipalmitoyl phosphatidylcholine;PC, phosphatidylcholine; PS, phosphatidyl serine; BR, bacteriorhodopsin.

The simplest single-celled organisms are composed of central regionsfilled with an aqueous material and a variety of soluble small moleculesand macromolecules. Enclosing this region is a membrane which iscomposed of phospholipids arranged in a bilayer structure. In morecomplex living cells, there are internal compartments and structuresthat are also enclosed by membranes. There are many protein moleculesembedded or associated within these membrane structures, and theseso-called membrane proteins are often the most important to determiningcell functions including communication and processing of information andenergy. The largest problem in studying membrane proteins is that theinside of the phospholipid bilayer is hydrophobic and the embedded oranchored part of the membrane protein is itself also hydrophobic. Inisolating these membrane proteins from their native membraneenvironments, it is very difficult to prevent them from forming inactiveaggregates. The present invention provides ways to generate a solublenanoparticle that is itself a native-like phospholipid bilayer intowhich hydrophobic proteins of interest (target proteins) can beincorporated to maintain the target protein as a soluble andmonodisperse entity. This is accomplished by incorporating hydrophobicproteins such as membrane proteins into nanometer scale structures.

Membrane Scaffold Proteins (MSPs) as used herein are artificial proteinswhich self assemble phospholipids and phospholipid mixtures intonanometer size membrane bilayers. A subset of these nanometer sizeassemblies are discoidal in shape, and are referred to as nanodisc ornanodisc structures. These structures preserve the overall bilayerstructure of normal membranes but provide a system which is both solublein solution and which can be assembled or affixed to a variety ofsurfaces.

The MSPs of the present invention must be amphipathic, with one part ofits structure more or less hydrophilic and facing the aqueous solventand the other part more or less hydrophobic and facing the center of thehydrophobic bilayer that is to be stabilized. Examination of the basicbiochemical literature reveals two candidates of protein structures thathave this required amphipathic character: the alpha-helix and thebeta-sheet. Although there are examples in the literature wherebeta-sheets can fold upon themselves to create a structure where the“inside” is hydrophobic and the “outside” is hydrophilic, the centralcavity so formed in the simplest of these structures is small. Such asmall internal region might stabilize a phospholipid bilayer, but thesize would be too small to incorporate any desired membrane proteintarget. Hence, we designed the MSPs of the present invention to have analpha helix as a fundamental amphipathic building block. Each MSP has anamino acid sequence which forms amphipathic □-helices with morehydrophobic residues (such as A, C, F, G, I, L, M, V, W or Y)predominantly on one face of the helix and more polar or chargedresidues (such as D, E, N, Q, S, T, H, K, or R) on the other face of thehelix. See FIG. 2 for a schematic representation. In addition, thehelical structure is punctuated with proline residues (P) about every20-25 amino acids to form “kinks” or to initiate turns to facilitate the“wrapping” of the MSP around the edge of a discoidal phospholipidbilayer. See FIG. 2, which depicts a generalized linear amino acidsequence and a helical wheel diagram showing the placement ofpredominantly hydrophobic amino acids on one face of the helix. Theexact amino acid sequence can vary in the positioning and number of thehydrophobic amino acids within the designed linear sequence. Simplemodels in which either the helical axis is parallel or perpendicular tothe normal of the Nanodisc bilayer can be generated; See FIGS. 3A and3B. To generate a disk with diameter of roughly 10 nm, an MSP comprisesabout 12 to about 20 or more repeating units having this generalizedamphipathic sequence. Preferably, this protein would be composed ofamphipathic alpha helices each with a length of between 14 and 25 aminoacids, punctuated in the linear sequence by a residue unfavorable forhelix formation, such as proline, which form small helical buildingblocks that stabilize the hydrophobic core of the phospholipid bilayer.These small helical segments are linked together with about 1 to about 5amino acid residues. To cover the edge of a 10 nm discoidal particle ineither the “belt” or “picket fence” models presented, one would needbetween 10-20 such helices, with 16 being the optimal number based onthe simple graphic analysis of FIGS. 3A and 3B. We thus built asynthetic gene to express a protein containing the desired amphipathichelices.

The MSPs of the present invention can be used to solubilize tethered,embedded or integral membrane proteins in nanoscale structures. Tetheredmembrane proteins are composed mostly of a relatively soluble globulardomain external to the bilayer and relatively simple (e.g., a singlepass helix) which anchors this domain to the membrane bilayer. Theglobular domain, in nature, can be extracellular or cytoplasmic inorientation. Embedded membrane proteins, as defined herein, are thosewhich include a membrane anchoring segment of the polypeptide, but whichalso have groupings of hydrophobic amino acids on the surface of theprotein, which hydrophobic domains are embedded within the membranebilayer. Integral membrane proteins are predominantly located within themembrane bilayer; relatively small portions of the protein are exposedto an aqueous environment within the cell or to the extracellularaqueous environment.

The tethered membrane protein class is exemplified by NADPH-cytochromeP450 reductase (e.g., from rat liver endoplasmic reticulum), cytochromeb5 and human tissue factor. NADPH-Cytochrome P450 reductase is amembrane protein found in the endoplasmic reticulum and catalyzespyridine nucleotide dehydration and electron transfer to membrane boundcytochrome P450s. Isozymes of similar structure are found in humans,plants, other mammals, insects etc. Tissue factor (TF), orthromboplastin, is a 30,000 Da type-I tethered membrane protein criticalto initiation of the blood coagulation cascade. This membrane-boundprotein acts as an activation cofactor for factor VII, the solubleserine protease which carries out the first enzymatic step in bloodcoagulation. Expression of tissue factor is limited to cells that arenot in direct contact with blood plasma, forming a “hemostatic envelope”around vasculature and the entire organism. High levels of TF are foundin the skin, brain, and the adventitial layer which surrounds bloodvessels. The TF:VII complex must be assembled on a membrane surface toexhibit high activity, and optimal activity is seen only when themembrane contains phospholipids with negatively charged headgroups.Cytochrome b5 is a membrane-anchored (tethered) heme protein having asingle membrane anchor domain that penetrates the membrane bilayer.Cytochrome b5 solubilized from its native membrane exists as largeaggregates in the absence of detergent and appears as a smear ratherthan a discrete band on native polyacrylamide gel electrophoresis (PAGE)(FIG. 17, lane 2). Formation of Nanodiscs through the self-assemblyprocess using MSPs taught in our invention, wherein cytochrome b5 isadded to the preparation of MSP and phospholipid results inincorporation of cytochrome b5 into disk-sized structures (lane 4 ofFIG. 17). This is verified by the intense heme-staining of the bandcorresponding to Nanodiscs in the right panel. Cytochrome b5-containingNanodiscs separated by anion-exchange chromatography are shown in lanes5 and 6 of FIG. 17. Two peaks elute from the anion exchange column near310 mM NaCl and near 370 mM NaCl. Disks alone were observed to elutenear 310 mM NaCl and cytochrome b5 alone to elute between 450 and 700 mMNaCl. This data demonstrates that cytochrome b5 is solubilized using theMSP technology and that disk complexes containing cytochrome b5 can bechromatographically separated and purified from undesired aggregatedmaterial. The optical absorption properties of the heme chromophore ofthe purified material show that the heme active site is in a nativeconformation.

Examples of embedded membrane proteins include, without limitation,cytochrome P450 2B4 from rabbit liver microsomes, cytochrome P450 3A4from human liver microsomes and cytochrome P450 6B1 from insectmicrosomes. The cytochromes P450 are a superfamily of enzymes that arefound in all forms of life. One role of many mammalian P450s is todetoxify xenobiotics; for instance, human liver P450s detoxify mostendogenous and exogenous compounds, and these enzymes determine the meanplasma lifetime of all drugs ingested. One of the most widely studiedhuman liver cytochrome P450s is cytochrome P450 3A4 (CYP 3A4). Thismembrane bound P450 is the most highly expressed P450 in human liver andis responsible for metabolizing almost 50% of all pharmaceuticals(Guengerich, F.P., Cytochrome P450. Cytochrome P450, ed. P. R. Ortiz deMontellano. 1995, New York: Plenum Press. 473-535.) In order todemonstrate the utility of Nanodisc technology for the study of thecytochromes P450, we incorporated CYP 3A4 into MSP supported nanobilayerdiscs. FIGS. 18-21 show that the retention times of the CYP 3A4(observed by optical absorbance at 417 nm) and the nanodiscs (monitoredat 280 nm where both MSP and P450 absorb) elute from the column at thesame time, approximately 24 minutes. This elution time also correlatesclosely to the calculated retention time of the disc protein complex.Further evidence that supports this is a native poly acrylamide gelelectrophoresis (PAGE) that directly measures the size of elutedNanodisc particles (FIG. 21).

Cytochrome P450 6B1 (CYP 6B1) is a member of the large cytochrome P450monooxygenase protein superfamily, and it is another example of anembedded membrane protein. CYP 6B1 has been isolated from Papiliopolyxenes, the black swallow tail, which feeds exclusively on plantsproducing furanocoumarins, plant metabolites that are phototoxic to mostorganisms. CYP 6B1 catalyzes the detoxification of furanocoumarins bywhat is believed to be an epoxidation reaction (Ma, R. et al. (1994)Arch. Biochem. Biophys. 310(2), 332-40). In order to show a newapplication of the MSP technology of the present invention, wedemonstrate that isolated membranes containing their repertoire ofmembrane proteins can be incorporated into nanodiscs comprising MSPs. Aparticularly important embodiment is the use of the common insect cellculture-baculovirus expression system which is used widely as aheterologous expression system. Thus, we used a commercially availableSf9 insect cell line co-infected such that a microsomal preparationcontaining overexpressed insect CYP 6B1 and an over-expressed insectNADPH cytochrome P450 reductase was produced. Hence, we not onlydemonstrated that MSP nanodiscs can be used to incorporate anothercytochrome P450 system into soluble monodisperse particles but also thatthe source of this P450 could be simply the whole membranes from the Sf9cell line that has been infected with a cloned CYP 6B1 gene. Thus, MSPsupported nanodiscs can be produced for use in high-throughput screeningventures such as the identification of ligands for membrane-associatedproteins and for the identification of new pharmaceuticals. Thisapplication can be extended to any other source of membrane fragmentscontaining target proteins of interest, such as any mammalian cellculture system or mammalian expression system.

An important utility of the nanodisc technology of the present inventionis in high throughput screening for enzymatic or receptor bindingactivity. In many such systems, it is advantageous to have more than onemembrane protein target incorporated into the nanodiscs, for example, ofthe electron transfer partner needed for P450 monooxygenase catalysis orthe corresponding G-protein incorporated with a G-protein coupledreceptor. In order to demonstrate the utility of the MSP nanodisctechnology in these situations, we successfully incorporated the NADPHcytochrome P450 reductase and a cytochrome P450 6B1. As demonstratedherein, each target membrane protein can be individually incorporatedinto nanodiscs using MSPs or they can be incorporated in combinations.The endogenous relative amounts of cytochrome P450 to reductase is about10-20 P450 molecules per reductase molecule (Feyereisen, R. (1999).Annual Review of Entomology 44, 501-533). To obtain activity of CYP6B1after reconstitution into disks, it is preferable that an excess amountof reductase be added to the reconstitution mixture, such that a P450molecule and reductase molecule both partition into a single disk.Supplementation of the microsomal preparation with exogenously addedreductase has been accomplished. The sample was separated by a Superdexsizing column, where absorbance at 280 nm indicates presence of MSP1,absorbance at 420 nm and 456 nm indicates presence of ferric species,and absorbance at 456 nm also indicates presence of reductase (FIG. 25).A ratio plot of 456 nm to 420 nm was made which showed positions on thechromatogram where the absorbance at 456 nm was above that associatedwith CYP6B1 and, therefore, could be attributed to absorbance byreductase (FIG. 26). Retention times correlated with the presence of 10nanometer particles containing both CYP6B1 and reductase. See also FIGS.22-24.

The integral membrane proteins are exemplified by the 7-helixtransmembrane proteins, including, but not limited to, bacteriorhodopsinfrom Halobacterium halobium, the 5-hydroxy tryptamine 1A G-proteincoupled receptor from Homo sapiens and other G-protein coupled proteinreceptors. Members of each class of membrane protein have beensuccessfully incorporated into the nanoscale structures using the MSPsand methods of the present invention. In particular, cell surfacereceptors, and especially G-protein coupled receptors, can beincorporated into nanobilayer bilayer structures formed from a class ofmembrane scaffold proteins (MSPs). BR has been incorporated into the MSPnanodiscs as described herein below, and we also used a commerciallyavailable insect cell expression system that provides a membranefraction hosting the G-protein coupled receptor human for 5-HT-1A(serotonin). The ligand binding activity documented for 5-HT-1Aincorporation into nanodiscs proves that the protein is in the activeconformation in the nanodiscs of the present invention.

FIG. 10, which shows a gel filtration elution profile of theMSP/BR/dimyristoyl phosphatidylcholine synthesis mixture, demonstratesthat bacteriorhodopsin has been solubilized. In the absence of MSP,bacteriorhodopsin elutes quantitatively in the void fractions. A majorpeak of bacteriorhodopsin elutes at a position slightly earlier than themajority of MSP disks (small complexes ˜100 Å in diameter, based onStoke's radii of calibration standards). Larger complexes containing BRare also formed. The BR present in the complexes can be specificallybound to and eluted from a nickel affinity column through an engineeredMSP that contains a 6His tag. In addition, the elution profile of BR ingel filtration is remarkably similar to elution profiles of MSP disks inthe absence of BR. The spectrum of BR in the complexes resembles that ofdetergent-solubilized monomeric BR and appears unchanged for severalweeks upon storage at 4° C. Surprisingly, MSP quantitatively solubilizesBR in the absence of exogenously added phospholipid. These lipid-poorcomplexes are also the size of small MSP/phospholipid disks.

We created an artificial MSP (MSP2) by designing a tandem repeat of MSP1connected by a short linker to create a new molecule. See FIG. 5G andSEQ ID NO:17. Relatively large quantities (tens of milligrams/liter cellculture) of the artificial MSPs of the present invention are produced ina bacterial expression system. Our constructs reduce the number of sizeclasses that can be formed (those corresponding to three MSP1molecules). Preliminary evidence indicates that the sizes of majorspecies formed with MSP2 correspond to two and four MSP1 molecules. Inaddition, the smallest disk sizes due to alternate conformations ofmembrane scaffold proteins that are found in preparations with MSP1 atlow phospholipid:MSP ratios do not exist for MSP2. Without wishing to bebound by theory, we believe that the smaller particles contain a singlemolecule of MSP2 and the larger disks contain two molecules.

The scaffold protein (MSP) has been engineered to minimize thevariability in the structure of the discoidal phospholipid bilayerentities, provide greater structural stability and increased sizehomogeneity of the disk structures, and to incorporate usefulfunctionalities such as tags for purification, and physical manipulationof disks. Disk homogeneity is necessary for efficient incorporation ofsingle membrane proteins or single membrane protein complexes into asingle size class of disk. The parent molecule, apo A-I, has functionsbeyond disk structure stabilization; these include cellular receptorbinding, LCAT activation and structural transformations between variouslipoprotein species (Forte et al., 1971; Holvoet et al., 1995; Fidge,1999). These functional regions are unnecessary and often deleterious inthe artificial bilayer systems of the present invention. The artificialscaffold protein can be used in studies of amphipathic helixmembrane-protein structures.

Secondary structure prediction provides a way of assessing structuralfeatures of the scaffold protein. The structure consists of mostlya-helix punctuated by prolines in the repeat sequences as shown in FIG.4. Eight to nine alpha helices are believed to associate with lipid inthe form of disks. The N-terminal region of apo A-I is predicted to bemore globular in character. This portion of the molecule has beenremoved to produce a construct that is capable of forming disks. An MSPthat produces disk assemblies with high monodispersity is desirable. Thecentral helices (99-186) can be displaced by the lipid-free form of arelated protein, apolipoprotein A-II, added to a solution of diskstructures (Durbin and Jonas, 1999), and that these helices can be partof a “hinge” domain (FIGS. 5A-5B) that is dissociated from the edge ofthe disk, producing various particle diameters within the LP2, LP3 andLP4 classes (Jonas et al., 1989). Disk forms with dissociated hingedregions are also more susceptible to proteolysis (Dalton and Swaney,1993), which is undesirable. The deletion of pairs of the centralhelices (100-143, 122-165, and 144-186) results in recombinants thatform disks of smaller size than full-length apo A-l, and in addition twoof the mutants (122-165 and 144-186) have increased stability tochemical denaturation (Frank et al., 1997). Further work replacedhelices 5 and 6 (143-186) with another set of helices (FIG. 5D). Helices3 and 4 contain regions that were thought to confer stability to disks(Frank et al., 1997). Helix 3 has a high lipid affinity and is believedto confer stability to the lipid-associated form through salt bridges.Helix 1 also has a high lipid affinity and is completely helical (Rogerset al., 1998). A construct which incorporates the helix 1-2 pair inplace of helix pair 5-6 is desirable. The roles of the half-repeats areof great interest. These 1 1-mer repeats are predicted to be α-helical,but are not long enough to span the bilayer in the picket fence model.In a molecular dynamics model of the disk in the picket fence model, theregion corresponding to half-repeat 1 was in fact “floppy” and did notinteract with lipid, while half-repeat 2 was found parallel to thebilayer near the headgroup region (FIGS. 5A and 5C). Such structures mayconfer disorder to the resulting disks. To ascertain the roles of halfrepeats and to further optimize the MSP structure and function,mutagenesis and directed evolution were used to generate variants thatare described herein below.

Receptors incorporated into MSP disks are useful in structural,biochemical and pharmaceutical research. Membrane protein study iscurrently limited to insoluble membrane dispersions, detergent micelles,and liposomes. Purified systems for biochemical and physical studyrequire stability, which may or may not obtainable with detergents inmany instances. Detergent micelles are dynamic and undergo structuralfluctuations that promote subunit dissociation and present difficulty inthe ability to handle proteins in dilute solution. The MSP nanobilayers(nanodiscs) are more robust structurally, having a phospholipid bilayermimetic domain of discrete size and composition, and greater stabilityand smaller surface area than unilamellar liposomes. The particles ofthe present invention are stable in size and structure for at least amonth at 4° C.

Signal transducing elements occur across membranes, while vesiclesrender one side of membrane in accessible to hydrophilic reagents andeffector proteins. A specific embodiment of the present invention usesdisks to stabilize pharmaceutical targets such as GPCRs, ion channels,receptor kinases, and phosphatases in a membrane-bound form on carrierparticles. We have incorporated proteins with multiple spanning helicesinto the disks of the present invention, with a focus on GPCRs. We havesuccessfully incorporated a model serpentine receptor,bacteriorhodopsin. Bacteriorhodopsin is a model for GPCRs, which arecurrent targets for drug discovery. Currently, over 1000 probableG-protein receptors from various organisms have been cloned and many ofthe so-called “orphan” receptors await identification of naturalligands. Ligand classes include peptide hormones, eicosanoids, lipids,calcium, nucleotides, and biogenic amines. GPCRs are believed to accountfor more than half of currently marketed pharmaceuticals. One ofordinary skill in the art can, without undue experiment, optimizemethods of incorporation of this structural class of membrane proteins.Structural characterization of the reconstituted receptors are performedusing chemical analysis, spectroscopy and atomic force microscopy asdescribed herein below.

The MSPs of the present invention are used in disks to solubilize,stabilize, and manipulate membrane proteins. The MSPs of the presentinvention, when formulated onto disks, are applicable in surfacetechnology such as biosensor chip for high throughput screening or solidphase assay techniques. Our work on disk scaffolds has also involvedsurface-associated assemblies. For instance, the SPR biosensor utilizesan approximately 50 nm thick gold film on an optical component to couplesurface plasmons to a dielectric component (sample) at the surface ofthe gold film. MSP stabilized bilayers can be attached to the surfacefor use as a biomimetic layer containing proteins or other targets ofinterest by engineering cysteines into the MSP (FIG. 15A). The use ofthiols is well known for attaching molecules to gold surfaces. Theplacement of the cysteine depends on the model used for placement of thecysteine residue(s). Based on the belt model, cysteines can be placedalong the polar side of the amphipathic helix axis, provided that acysteine residue is not positioned at the helix-helix interface. Thehelix-helix interface of the belt is believed to be in register with theposition of apo A-I Milano (R173C), which forms disulfide-linked dimers(Segrest et al., 1999). An alternative is to introduce cysteines withina flexible N- or C-terminal linker. Such a construct is, in theory,capable of associating either the belt or the picket fence form of diskto a gold surface. Alternatively, thiol lipids can be incorporatedwithin the bilayer domain. In addition to SPR, surface-associated diskson gold can be used in STM and electrochemical studies, for example,such as with membrane associated redox proteins, e.g. cytochrome P450and its flavoprotein, as well as ion channels.

SPR data can also be obtained from measurements made using a thin filmof dielectric such as silicon dioxide applied over the metal filmnormally used as the substrate in SPR. This variation of the techniquehas been termed coupled plasmon waveguide resonance (CPWR) (Salamon etal., 1997a). Because silica can be used as the active surface in theseplasmon resonance experiments, the process of producing a self-assembledbilayer can be simplified to the same procedures used to producesurfaces on mica or other silicon oxide surfaces. This has the addedadvantage of making the conditions used for the SPR experiments directlycomparable to those used for AFM experiments. The CPWR technique caneasily be performed on our SPR instrument by simply adding the silicacoating to the metal film slides that are presently used for SPRspectroscopy.

MSPs with available cysteine groups also enable specific labeling withchemically reactive groups or affinity tags for immobilization in gelmatrices. Hydrogels with reactive coupling groups are useful forimmobilizing proteins for SPR measurements. In the hydrogelconfiguration (FIG. 15B), the disk would serve as a carrier forbilayer-embedded membrane proteins in a monodisperse form with bothintra- and extracellular domains available for ligand binding. We havealready demonstrated that disks containing His tag bind to a metalchelate matrix which can be used to immobilize BR. This points toanother use of the disk structure, i.e., in preparing affinity matricesfor bioseparation processes, and measuring ligand affinities. Theparticles and techniques with the present invention are useful for drugdiscovery, structure/function correlation, and structure determinationof membrane proteins.

Current limitations to structure determination of membrane proteins arethe abilities to produce large amounts of membrane proteins, and tocrystallize these proteins. MSPs are useful as carriers for membraneprotein stabilization and expression. MSP can serve to solubilizemembrane proteins for crystallization in lieu of detergents. Indeed,where the lipid bound form of MSP is structurally stable and rigid,crystallization is enhanced by introduction of crystal contacts throughthe MSP. We have already demonstrated that MSP1 or MSP2 can be used tosolubilize BR in the presence and absence of exogenous lipid. Additionalnonexemplified fusion constructs with membrane protein with an MSPregion can be expressed in Escherichia coli using any of a number ofart-known vectors. In this way, a stable and soluble form of themembrane protein that contains a membrane anchor is produced in largequantity. The exciting discovery that MSP solubilizes BR in the absenceof added phospholipid allows the use of the artificial MSP to stabilizemembrane proteins in the absence of detergents or lipid additives. Theartificial MSPs of the present invention can be used in solubilizationof BR and other membrane proteins including, but not limited to,cytochrome P450, cytochrome P450 reductase, and the 5-HT-1A receptor.

One important goal in utilizing a membrane scaffold protein (MSP) toprovide membrane proteins in general, and G-protein Coupled Receptors(GPCRs) in particular, with a suitable environment for homogeneousbiochemical assay or crystallization is to have a homogeneouspreparation of particles. The membrane scaffold proteins we havedescribed, ranging from full length human Apo-AI and its derivatives, toalternate membrane scaffold proteins including but not limited to,truncated human Apo-AI (t-MSP) where the amino terminal soluble domainhas been removed, deletion mutants where one or more protein segmentsare removed and any of the above materials where a histidine tag isincorporated, primarily form 8-10 nm particles when self-assembled withphospholipids in solution. However, upon initial reconstitution, thereare particles of other sizes present. While standard size separationchromatography can be used to purify a single size class of particle, itis preferable to minimize the size distribution of the initialreconstitution mixture of target protein, MSP and phospholipid. The 8-10nm particle is nominally composed of two MSP, Apo-AI or Apo-AIderivative proteins. Hence, we constructed a membrane scaffold proteinwhere two of the truncated Apo-AI proteins (termed MSP1) are geneticallyliked to form a scaffold protein composed of a single polypeptide chain.This is schematically illustrated in FIG. 5G.

GPCRs which can be solubilized in nanoscale phospholipid bilayers caninclude the Class A (Rhodopsin-like ) GPCRs, which bind amines,peptides, hormone proteins, rhodopsin, olfactory prostanoid,nucleotide-like compounds, cannabinoids, platelet activating factor,gonadotropin-releasing hormone, thyrotropin-releasing hormone andsecretagogue, melatonin and lysosphingolipid and LPA. GPCRs with amineligands include, without limitation, acetylcholine or muscarinic,adrenoceptors, dopamine, histamine, serotonin or octopamine receptors);peptide ligands include but are not limited to angiotensin, bombesin,bradykinin, anaphylatoxin, Fmet-leu-phe, interleukin-8, chemokine,cholecystokinin, endothelin, melanocortin, neuropeptide Y, neurotensin,opioid, somatostatin, tachykinin, thrombin vasopressin-like, galanin,proteinase activated, orexin and neuropeptide FF, adrenomedullin (G10D),GPR37/endothelin B-like, chemokine receptor-like and neuromedin U.

Ligands of other specific GPCRs include hormone protein, rhodopsin,olfactory, prostanoid, nucleotide-like (adenosine, purinoceptors),cannabinoid, platelet activating factor, gonadotropin-releasing hormone,thyrotropin-releasing hormone & secretagogue, melatonin andlysosphingolipid & LPA, among others. Class B secretin-like GPCRsinclude, without limitation, those which bind calcitonin, corticotropinreleasing factor, gastric inhibitory peptide, glucagon, growthhormone-releasing hormone, parathyroid hormone, PACAP, secretin,vasoactive intestinal polypeptide, diuretic hormone, EMR1 andlatrophilin. Class C metabotropic glutamate receptors include thosewhich bind metabotropic glutamate, extracellular calcium-sensing orGABA-B, among others. “Orphan” receptors whose ligands are not yet knownare also potential targets of assays of the present invention.

In the assays of the present invention which demonstrate binding of aparticular ligand or which are used to identify inhibitors orcompetitors of ligand binding to an MSP-supported GPCR, a variety oflabels can be incorporated within the ligand molecule (such asradioactive isotope, e.g., ³H, ¹⁴C, ³⁵S, ³²P) or detectable compoundscan be attached to the ligand molecule provided that binding to thecognate receptor is not significantly reduced due to the label. Labelscan include, without limitation, ¹²⁵I, ¹³¹I, fluorescent compounds,luminescent compounds, etc).

The necessary properties of the linker sequence between fused MSPs areflexibility and solubility so that the fused proteins assemble intoparticles in a manner similar to two separate MSP. Linker sequencesconsisting of repeats of Gly-Gly-Gly-Ser/Thr-(SEQ ID NO:46) have theseproperties. It is also desirable to minimize the length of the linker.We constructed a fusion with the minimal linker -GT-, which correspondsto the consensus DNA restriction site for Kpn I, as described hereinbelow. The Kpn I site provides an easy way of inserting any desiredlinker sequence by restriction with Kpn I and insertion ofdouble-stranded synthetic DNA encoding any desired linker (Robinson etal. 1998). We have also made a fusion construct with the linker sequence-GTGGGSGGGT-(SEQ ID NO:15). The MSP2 with the minimal linker, however,assembles into particles very similar to particles containing two MSP1proteins.

The complete amino acid and nucleic acid sequences for the MSP2 scaffoldprotein is shown in Tables 7 and 8; see also SEQ ID NO:16 and SEQ IDNO:17. The MSP2 fusion protein was expressed in E. coli and purified tohomogeneity using basically the same procedure as described for thesingle MSPs. The MSP2 protein serves as an effective scaffold protein,self-assembling with phospholipid upon removal of solubilizingdetergent. Addressing the point of sample heterogeneity, FIG. 13 showsdensitometry traces of native gradient polyacrylamide gels with theindividual peaks labeled as to mean particle diameter in Angstroms.Clearly evident, particularly at a lipid/dimer ratio of 200corresponding to nominally 10 nm particles, is the much greatermonodispersivity afforded by the MSP2 protein.

Importantly, the overall stability of the disks, as monitored bychemically induced unfolding and exposure of tryptophan residues tosolvent, is not altered by the fusion of the monomeric membrane scaffoldproteins, as shown in FIG. 14.

An important technique used in the characterization of disk structuresand associated proteins is scanning probe microscopy (SPM). SPM is anumbrella term for any microscope that utilizes the scanning principlesfirst pioneered in the scanning tunneling microscope (STM), but thesemicroscopes can vary so greatly they are best discussed in terms oftheir guiding central principle. The technology has been used in theanalysis of biological membranes and their associated proteins, bilayerstructures and incorporated membrane proteins surfaces. SPM combinesindependent mobility in all three spatial directions (scanning) with adetection system capable of detecting some characteristic of the surface(probing). The various surface characteristics that can be probed(conductivity, surface forces, compressibility, capacitance, magnetic,fluorescence emission) demonstrate the wealth of information that can beobtained. The excellent z-axis sensitivity of atomic force microscopymakes the presence of proteins binding to an rHDL monolayer easilydetectable, as we have shown for well characterized integral andanchored membrane proteins immobilized in MSP-supported nanodiscs(Bayburt et al., 1998). Other examples of the usefulness of the preciseheight measurements possible with AFM are our direct quantitation ofrHDL particle height (Carlson et al., 1997), and membrane protein heightmeasurements obtained by modulating the force of the AFM probe onvarious nanodisc assemblies (Bayburt et al., 2000). The surfaceassociation of disks formed from MSPs allows the utilization of thehuman apo A-I protein and its improved variants to directly investigatethe biophysical properties of single membrane proteins incorporated intophospholipid bilayers on surfaces by SPM. The ability to attach disks toatomically flat conductive surfaces (such as gold) is necessary forscanning tunneling microscopy (STM). In theory, tunneling through aredox-active system can be used to probe the functional state of anenzyme (Friis et al., 1999; Mukhopadhyay et al., 2000). These twotechniques provide complementary data and can be used in concert tocreate as complete a picture as possible of events occurring at thebilayer/solution interface. The ability to place disks on a gold surfacealso allows the use of surface plasmon resonance (SPR). Insertion ofmembrane proteins into such artificial lipid bilayers, or theirinteraction with surface-associated proteins can be detected andquantified by SPR.

Measurements of disk stabilities and determination of size dispersionamong classes are necessary to evaluate the constructs that are beingcreated. Gel filtration and native gel electrophoresis are used toseparate and quantitate the different size classes. Spectroscopy is usedto quantitate secondary structure (CD) and lipid association(fluorescence) characteristics of the engineered MSPs, includingstabilities based on thermal and chemical denaturation. Compositions andstoichiometries of components in disks are quantitated by traditionalmethods (Jonas, 1986).

AFM is used to provide molecular resolution data on the structuralorganizations of the lipid and protein components of the systemsproduced. This technique can be used in air, vacuum, and under aqueousand non-aqueous fluids. The latter capability has made it the mostimportant scanning probe technique in the biological sciences. The AFMis a very versatile instrument as it is capable of acquiring images andother forms of force data in several different modes (Sarid, 1994) suchas contact, tapping, phase, and lateral force. All of these scanningmodes are available on the Digital Instruments Multimode Scanning ProbeMicroscope (Digital Instruments, Plainview, N.Y.) and they have beensuccessfully used to image rHDL and proteins associated with rHDL layersunder biological buffers. This instrument can also be used in STM andelectrochemical modes to study characteristics of gold-associatedconstructs and incorporated redox proteins.

As used herein, membrane scaffold proteins are proteins or polypeptideswhich self assemble phospholipids and phospholipid mixtures intonanometer size membrane bilayers. A subset of these structures arediscoidal in shape and are referred to as nanodiscs or nanodisks.Hydrophobic proteins, e.g., membrane proteins, or membrane fragments canassociate with these particles such that the hydrophobic proteins ormembrane fragments are effectively solubilized in a stable structurewhich maintains the functionality of the protein with respect toenzymatic activity or ligand binding. These particles are stable insolution or they can be fixed to a surface, advantageously in a uniformorientation with respect to the surface. As used herein, a nanoparticlecomprising MSPs (with or without another hydrophobic or a partiallyhydrophobic protein) can be from about 5 to about 500 nm, desirablyabout 5 to about 100 nm, or about 5 to about 50 nm in diameter.Nanoparticles (disks) of about 5 to about 15 nm in diameter areespecially useful.

We have shown that both MSP1 and MSP2 assemble with bacteriorhodopsin.From the initial reconstitution mixture, twobacteriorhodopsin-containing species are observed when particles areformed with MSP1 (FIG. 11) or MSP2 (FIG. 12) in the absence of addedphospholipid. We found that the MSP is absolutely required for thesolubilization of bacteriorhodopsin to form these species becauseomission of an MSP from the formation mixture results in largenon-specific bacteriorhodopsin aggregates that elute in the void volumeof the gel filtration column. The small peak at 15 minutes in FIG. 11represents BR aggregates. In these experiments, it appears that themajority of bacteriorhodopsin appears solubilized in the presence ofMSPs. The two sizes of particles observed are completely consistent withthe putative “hinge domain” adopting alternate conformations in thestructures. From previous work, this flexible hinge region is believedto consist of helices corresponding to helices 5 and 6 of human Apo-AIand thus by inference to MSP1. Thus, in the 9.8 nanometer diameterbacteriorhodopsin-containing particles, these flexible parts of theprotein structure appear to be associated with the hydrophobic core ofthe structure while in 7.6 nanometer diameter particles, this helicalregion is dissociated from the hydrophobic core, thus forming a smallerdiameter particle.

Further modifications of the parent Apo-AI protein can generate moreeffective and stable membrane scaffold proteins. For instance, toincrease the homogeneity of the BR/MSP structures and to address theissue of the flexible “hinge” region of the protein structure discussedabove, we have deleted the hinge domain region to produce two newmembrane scaffold proteins. In the first case, putative helical regions4 and 5 were deleted from the MSP1 histidine-tagged construct to producea construct called MSP1 D5-6. In a second experiment, the putativehelices 5 and 6 were deleted to produce a material termed MSP1 D4-5. Wehave overexpressed these proteins in E. coli, which are expressed athigh levels upon induction of expression withisopropyl-thio-b-D-galactopyranoside in lac-regulated constructs.

An alternative way of avoiding the formation of multiple particle sizeclasses is to engineer MSP constructs so that the hinge domain helicesare replaced by helices having higher affinities for the hydrophobiccore of the particles. In this case, the higher affinity interactiondisfavors the formation of the smaller species wherein the hinge domainis dissociated. In this experimental line, we have chosen to replace thehinge region (helices 5 and 6) with the protein sequence correspondingto the native sequence corresponding to helices 1 and 2. In anothermanifestation, we have chosen a DNA construct encoding a membranescaffold protein wherein the protein sequence corresponding to theputative helical regions 3 and 4 are used to replace the hinge regionwith a goal of yielding a single size particle upon assembly withbacteriorhodopsin and lipid.

The so-called “half-repeat” units present in the parent human Apo-AIprotein also may give rise to conformational heterogeneity in MSPassemblies. For instance, in the picket fence model these helices adopta conformation parallel to the bilayer plane and do not play a majorrole in interactions with the hydrophobic core of the particle as theother regions of the protein sequence are envisioned to contribute. Inthe “belt model” these short helical repeats could give rise tosegmented mobility allowing the MSP to adopt different conformations. Inother words, a MSP in which the number of types of structural elementsis minimized is most likely desirable embodiment of the membranescaffold protein concept. Thus, in order to further optimize thestructure of the membrane scaffold protein with respect to their abilityto solubilize integral membrane protein targets, we can engineerderivative sequences that will delete both half-repeat units to producea simplified MSP structure.

Monoclonal or polyclonal antibodies, preferably monoclonal, specificallyreacting with an MSP of the present invention can be made by methodsknown in the art. See, e.g., Harlow and Lane (1988) Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratories; Goding (1986)Monoclonal Antibodies: Principles and Practice, 2d ed., Academic Press,New York; and Ausubel et al. (1993) Current Protocols in MolecularBiology, Wiley Interscience, New York, N.Y.

Standard techniques for cloning, DNA isolation, amplification andpurification, for enzymatic reactions involving DNA ligase, DNApolymerase, restriction endonucleases and the like, and variousseparation techniques are those known and commonly employed by thoseskilled in the art. A number of standard techniques are described inSambrook et al. (1989) Molecular Cloning, Second Edition, Cold SpringHarbor Laboratory, Plainview, New York; Maniatis et al. (1982) MolecularCloning, Cold Spring Harbor Laboratory, Plainview, New York; Wu (ed.)(1993) Meth. Enzymol. 218, Part I; Wu (ed.) (1979) Meth. Enzymol. 68; Wuet al. (eds.) (1983) Meth. Enzymol. 100 and 101; Grossman and Moldave(eds.) Meth. Enzymol. 65; Miller (ed.) (1972) Experiments in MolecularGenetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York;Old and Primrose (1981) Principles of Gene Manipulation, University ofCalifornia Press, Berkeley; Schleif and Wensink (1982) Practical Methodsin Molecular Biology; Glover (ed.) (1985) DNA Cloning Vol. I and II, IRLPress, Oxford, UK; Hames and Higgins (eds.) (1985) Nucleic AcidHybridization, IRL Press, Oxford, UK; Setlow and Hollaender (1979)Genetic Engineering: Principles and Methods, Vols. 1-4, Plenum Press,New York; and Ausubel et al. (1992) Current Protocols in MolecularBiology, Greene/Wiley, New York, N.Y. Abbreviations and nomenclature,where employed, are deemed standard in the field and commonly used inprofessional journals such as those cited herein.

All references cited in the present application are incorporated byreference herein to the extent that there is no inconsistency with thepresent disclosure.

The description provided herein is not intended to limit the scope ofthe invention as claimed herein. Any variations in the exemplifiedarticles and methods which occur to the skilled artisan are intended tofall within the scope of the present invention.

EXAMPLES Example 1 Construction of Recombinant DNA Molecules forExpression of MSPs

The human proapo A-I coding sequence as given below was inserted betweenNco I and Hind III sites (underlined) in pET-28 (Novagen, Madison,Wis.). Start and stop codons are in bold type. The restrictionendonuclease recognition sites used in cloning are underlined. TABLE 1ProApoAl coding sequence (SEQ ID NO:1) Restriction sites used in cloningare underlined, and the translation start and stop signals are shown inbold. CCATGGCCCATTTCTGGCAGCAAGATGAACCCCCCCAGAGCCCCTGGGATCGAGTGAAGGACCTGGCCACTGTGTACGTGGATGTGCTCAAAGACAGCGGCAGAGACTATGTGTCCCAGTTTGAAGGCTCCGCCTTGGGAAAACAGCTAAACCTAAAGCTCCTTGACAACTGGGACAGCGTGACCTCCACCTTCAGCAAGCTGCGCGAACAGCTCGGCCCTGTGACCCAGGAGTTCTGGGATAACCTGGAAAAGGAGACAGAGGGCCTGAGGCAAGAGATGAGCAAGGATCTGGAGGAGGTGAAGGCCAAGGTGCAGCCCTACCTGGACGACTTCCAGAAGAAGTGGCAGGAGGAGATGGAGCTCTACCGCCAGAAGGTGGAGCCGCTGCGCGCAGAGCTCCAAGAGGGCGCGCGCCAGAAGCTGCACGAGCTGCAAGAGAAGCTGAGCCCACTGGGCGAGGAGATGCGCGACCGCGCGCGCGCCCATGTGGACGCGCTGCGCACGCATCTGGCCCCCTACAGCGACGAGCTGCGCCAGCGCTTGGCCGCGCGCCTTGAGGCTCTCAAGGAGAACGGCGGCGCCAGACTGGCCGAGTACCACGCCAAGGCCACCGAGCATCTGAGCACGCTCAGCGAGAAGGCCAAGCCCGCGCTCGAGGACCTCCGCCAAGGCCTGCTGCCCGTGCTGGAGAGCTTCAAGGTCAGCTTCCTGAGCGCTCTCGAGGAGTACACTAAGAAGCTCAACACCC AGTAATAAGCTT-3′

TABLE 2 Proapoal amino acid sequence (SEQ ID NO:2)MAHFWQQDEPPQSPWDRVKDLATVYVDVLKDSGRDYVSQFEGSALGKQLNLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ

The construction of the MSP1 coding sequence was accomplished asfollows. Primers were designed to produce DNA encoding MSP1, thetruncated protein lacking the N-terminal domain of proApoAl, bypolymerase chain reaction (PCR) mutagenesis (Higuchi et al., 1988).

-   Primer 1 (SEQ ID NO:3)-   5′-TATACCATGGGCCATCATCATCATCATCATATAGMGGAAGACTAAAGCTCCTTG ACAACT-3′)    introduces an N-terminal 6-histidine tag for purification and    manipulation of MSP1, and a factor Xa cleavage site for removal of    the histidine tag. Factor Xa cleaves after R in the protein sequence    IEGR.

Primer 2 (SEQ ID NO:4) (5′-GCAAGCTTATTACTGGGTGTTGAGCTTCTT-3′) was usedas a reverse primer. TABLE 3 Histidine-tagged MSP1 coding sequence (SEQID NO:5). Restriction sites used in cloning are underlined, and thetranslation start and stop signals are shown in bold.TATACCATGGGCCATCATCATCATCATCATATAGAAGGAAGACTAAAGCTCCTTGACAACTGGGACAGCGTGACCTCCACCTTCAGCAAGCTGCGCGAACAGCTCGGCCCTGTGACCCAGGAGTTCTGGGATAACCTGGAAAAGGAGACAGAGGGCCTGAGGCAGGAGATGAGCAAGGATCTGGAGGAGGTGAAGGCCAAGGTGCAGCCCTACCTGGACGACTTCCAGAAGAAGTGGCAGGAGGAGATGGAGCTCTACCGCCAGAAGGTGGAGCCGCTGCGCGCAGAGCTCCAAGAGGGCGCGCGCCAGAAGCTGCACGAGCTGCAAGAGAAGTTGAGCCCACTGGGCGAGGAGATGCGCGACCGCGCGCGCGCCCATGTGGACGCGCTGCGCACGCATCTGGCCCCCTACAGCGACGAGCTGCGCCAGCGCTTGGCCGCGCGCCTTGAGGCTCTCAAGGAGAACGGCGGCGCCAGACTGGCCGAGTACCACGCCAAGGCCACCGAGCATCTGAGCACGCTCAGCGAGAAGGCCAAACCCGCGCTCGAGGACCTCCGCCAAGGCCTGCTGCCCGTGCTGGAGAGCTTCAAGGTCAGCTTCCTGAGCGCTCTCGAGGAGTACACTAAGAAGCTCAACACCCAGTAATAAGC TTGC

TABLE 4 Histidine-tagged MSP1 amino acid sequence (SEQ ID NO:6)MGHHHHHHIEGRLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLS ALEEYTKKLNTQ

For production of MSP1 without a N-terminal histidine tag, primer 1 wasreplaced with primer 1a: 5′-TACCATGGCAAAGCTCCTTGACAACTG-3′ (SEQ ID NO:7)to produce the sequence provided in SEQ ID NO:8. TABLE 5Non-Histidine-tagged MSP1 DNA sequence (SEQ ID NO:8). Restriction sitesused in cloning are underlined, and the translation start and stopsignals are shown in bold.TACCATGGCAAAGCTCCTTGACAACTGGGACAGCGTGACCTCCACCTTCAGCAAGCTGCGCGAACAGCTCGGCCCTGTGACCCAGGAGTTCTGGGATAACCTGGAAAAGGAGACAGAGGGCCTGAGGCAGGAGATGAGCAAGGATCTGGAGGAGGTGAAGGCCAAGGTGGAGCCCTACCTGGACGACTTCCAGAAGAAGTGGCAGGAGGAGATGGAGCTCTACCGCCAGAAGGTGGAGCCGCTGCGCGCAGAGCTCCAAGAGGGCGCGCGCCAGAAGCTGCACGAGCTGCAAGAGAAGTTGAGCCCACTGGGCGAGGAGATGCGCGACCGCGCGCGCGCCCATGTGGACGCGCTGCGCACGCATCTGGCCCCCTACAGCGACGAGCTGCGCCAGCGCTTGGCCGCGCGCCTTGAGGCTCTCAAGGAGAACGGCGGCGCCAGACTGGCCGAGTACGACGCCAAGGCCACCGAGCATCTGAGCACGCTCAGCGAGAAGGCCAAACCCGCGCTCGAGGACCTCCGCCAAGGCCTGCTGCCCGTGCTGGAGAGCTTCAAGGTCAGCTTGCTGAGCGCTCTCGAGGAGTACACTAAGAAGCTCAA CACCCAGTAATAAGCTTGC

TABLE 6 Non-Histidine-tagged MSP1 amino acid sequence (SEQ ID NO:9).MAKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNT Q

The production of an MSP with tandem repeats (MSP2) was carried at asdescribed below. The following primers were used to generate MSP2 (seeFIGS. 6A-6B): Primer 3 (SEQ ID NO:10): 5′-TACCATGGCAAAGCTCCTTGACAACTG-3′Primer3a (SEQ ID NO:11):5′-TATACCATGGGCCATCATCATCATCATCATATAGAAGGAAGACTAAA GCTCCTTGACAACT-3′Primer 4 (SEQ ID NO:12):5′-TAAGAAGCTCAACACCCAGGGTACCGGTGGAGGTAGTGGAGGTGGTA CCCTA-3 Primer 5 (SEQID NO:13): 5′-CAGGGTACCGGTGGAGGTAGTGGAGGTGGTACCCTAAAGCTCCTTGA CAA-3′Primer 6 (SEQ ID NO:14): 5′-GCAAGCTTATTACTGGGTGTTGAGCTTCTT-3′

In a first PCR, primer 2 (or primer 2a for N-terminal histidine tag) andprimer 4 were used to add a linker (encoding the amino acid sequenceGTGGGSGGGT; SEQ ID NO:15) to the 3′ end of the MSP gene to produceMSP-A. In a second PCR, the linker was added to the 5′ end of the MSPgene to produce MSP-B. Treatment of MSP-A and MSP-B with KpnI andsubsequent ligation produced the following constructs, one with and onewithout the linker. The Kpn I site provides an easy way to inserting anydesired linker sequence by restriction with Kpn I and religation withdouble-stranded synthetic DNA encoding desired linker. See FIGS. 7A-7B.TABLE 7 MSP2 (with histidine tag, without long linker) DNA sequence (SEQID NO:16). The translation start and stop codons are in bold type, andthe restriction endonuclease recognition sites used in cloning areunderlined. TATACCATGGGCCATCATCATCATCATCATATAGAAGGAAGACTAAAGCTCCTTGACAACTGGGACAGCGTGACCTCCACCTTCAGCAAGCTGCGCGAACAGCTCGGCCCTGTGACCCAGGAGTTCTGGGATAACCTGGAAAAGGAGACAGAGGGCCTGAGGCAGGAGATGAGCAAGGATCTGGAGGAGGTGAAGGCCAAGGTGCAGCCCTACCTGGACGACTTCCAGAAGAAGTGGCAGGAGGAGATGGAGCTCTACCGCCAGAAGGTGGAGCCGCTGCGCGCAGAGCTCCAAGAGGGCGCGCGCCAGAAGCTGCACGAGCTGCAAGAGAAGCTGAGCCCACTGGGCGAGGAGATGCGCGACCGCGCGCGCGCCCATGTGGACGCGCTGCGCACGCATCTGGCCCCCTACAGCGACGAGCTGCGCCAGCGCTTGGCCGCGCGCCTTGAGGCTCTCAAGGAGAACGGCGGCGCCAGACTGGCCGAGTACCACGCCAAGGCCACCGAGCATCTGAGCACGCTCAGCGAGAAGGCCAAGCCCGCGCTCGAGGACCTCCGCCAAGGCCTGCTGCCCGTGCTGGAGAGCTTCAAGGTCAGCTTCCTGAGCGCTCTCGAGGAGTACACTAAGAAGCTCAACACCCAGGGTACCCTAAAGCTCCTTGACAACTGGGACAGCGTGACCTCCACCTTCAGCAAGCTGCGCGAACAGCTCGGCCCTGTGACCCAGGAGTTCTGGGATAACCTGGAAAAGGAGACAGAGGGCCTGAGGCAGGAGATGAGCAAGGATCTGGAGGAGGTGAAGGCCAAGGTGCAGCCCTACCTGGACGACTTCCAGAAGAAGTGGCAGGAGGAGATGGAGCTCTACCGCCAGAAGGTGGAGCCGCTGCGCGCAGAGCTCCAAGAGGGCGCGCGCCAGAAGCTGCACGAGCTGCAAGAGAAGCTGAGCCCACTGGGCGAGGAGATGCGCGACCGCGCGCGCGCCCATGTGGACGCGCTGCGCACGCATCTGGCCCCCTACAGCGACGAGCTGCGCCAGCGCTTGGCCGCGCGCCTTGAGGCTCTCAAGGAGAACGGCGGCGCCAGACTGGCCGAGTACCACGCCAAGGCCACCGAGCATCTGAGCACGCTCAGCGAGAAGGCCAAGCCCGCGCTCGAGGACCTCCGCCAAGGCCTGCTGCCCGTGCTGGAGAGCTTCAAGGTCAGCTTCCTGAGCGCTCTCGAGGAGTACACTAAGAAGCTCAACACCCAGTA ATAAGCTTGC

TABLE 8 MSP2 (with histidine tag, without long linker) amino acidsequence (SEQ ID NO:17)MGHHHHHHIEGRLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMS/KDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQGTLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVS FLSALEEYTKKLNTQ

TABLE 9 MSP2 (with histidine tag, with long linker) DNA sequence (SEQ IDNO:18). Translation start and stop codons are in bold type; restrictionendonuclease sites used in cloning are underlined.TACCATGGGCCATCATCATCATCATCATATAGAAGGAAGACTAAAGCTCCTTGACAACTGGGACAGCGTGACCTCCACCTTCAGCAAGCTGCGCGAACAGCTCGGCCCTGTGACCCAGGAGTTCTGGGATAACCTGGAAAAGGAGACAGAGGGCCTGAGGCAGGAGATGAGCAAGGATCTGGAGGAGGTGAAGGCCAAGGTGCAGCCCTACCTGGACGACTTCCAGAAGAAGTGGCAGGAGGAGATGGAGCTCTACCGCCAGAAGGTGGAGCCGCTGCGCGCAGAGCTCCAAGAGGGCGCGCGCCAGAAGCTGCACGAGCTGCAAGAGAAGCTGAGCCCACTGGGCGAGGAGATGCGCGACCGCGCGCGCGCCCATGTGGACGCGCTGCGCACGCATCTGGCCCCCTACAGCGACGAGCTGCGCCAGCGCTTGGCCGCGCGCCTTGAGGCTCTCAAGGAGAACGGCGGCGCCAGACTGGCCGAGTACCACGCCAAGGCCACCGAGCATCTGAGCACGCTCAGCGAGAAGGCCAAGCCCGCGCTCGAGGACCTCCGCCAAGGCCTGCTGCCCGTGCTGGAGAGCTTCAAGGTCAGCTTCCTGAGCGCTCTCGAGGAGTACACTAAGAAGCTCAACACCCAGGGTACCGGTGGAGGTAGTGGAGGTGGTACCCTAAAGCTCCTTGACAACTGGGACAGCGTGACCTCCACCTTCAGCAAGCTGCGCGAACAGCTCGGCCCTGTGACCCAGGAGTTCTGGGATAACCTGGAAAAGGAGACAGAGGGCCTGAGGCAGGAGATGAGCAAGGATCTGGAGGAGGTGAAGGCCAAGGTGCAGCCCTACCTGGACGACTTCCAGAAGAAGTGGCAGGAGGAGATGGAGCTCTACCGCCAGAAGGTGGAGCCGCTGCGCGCAGAGCTCCAAGAGGGCGCGCGCCAGAAGCTGCACGAGCTGCAAGAGAAGCTGAGCCCACTGGGCGAGGAGATGCGCGACCGCGCGCGCGCCCATGTGGACGCGCTGCGCACGCATCTGGCCCCCTACAGCGACGAGCTGCGCCAGCGCTTGGCCGCGCGCCTTGAGGCTCTCAAGGAGAACGGCGGCGCCAGACTGGCCGAGTACCACGCCAAGGCCACCGAGCATCTGAGCACGCTCAGCGAGAAGGCCAAGCCCGCGCTCGAGGACCTCCGCCAAGGCCTGCTGCCCGTGCTGGAGAGCTTCAAGGTCAGCTTCCTGAGCGCTCTCGAGGAGTACACTAAGAAGCTCAACACCCAGTAATAAGCTTGC

TABLE 10 MSP2 (with histidine tag, with long linker, in bold type) aminoacid sequence (SEQ ID NO:19).MGHHHHHHIEGRLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQGTGGGSGGGTLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ

To delete hinge regions, deletion of helices 4 and 5 was carried out byconstructing the C-terminal portion of MSP1 using the following PCRprimers and the Sac I and Hind III fragment of the MSP1 coding sequenceas template. Primer A (SEQ ID NO:20):5′-TGGAGCTCTACCGCCAGAAGGTGGAGCCCTACAGCGACGAGCT-3′/ Primer B (SEQ IDNO:21): 5′-GCAAGCTTATTACTGGGTGTTGAGCTTCTT-3′.

This amplification product was digested with SacI and HindIII andligated into pLitmus 28 for sequencing. The Sac I+HindIII treatedhistidine-tagged MSP1 construct in pET 28 vector was then ligated withthe above fragment to produce MSP1 D4-5. TABLE 11 MSP1D4-5 (helices 4and 5 deleted) DNA sequence (SEQ ID NO:22). Translations start and stopcodons are in bold type; restriction endonuclease recognition sites areunderlined. TATACCATGGGCCATCATCATCATCATCATATAGAAGGAAGACTAAAGCTCCTTGACAACTGGGACAGCGTGACCTCCACCTTCAGCAAGCTGCGCGAACAGCTCGGCCCTGTGACCCAGGAGTTCTGGGATAACCTGGAAAAGGAGACAGAGGGCCTGAGGCAGGAGATGAGCAAGGATCTGGAGGAGGTGAAGGCCAAGGTGCAGCCCTACCTGGACGACTTCCAGAAGAAGTGGCAGGAGGAGATGGAGCTctaccgccagaaggtggagcCCTACAGCGACGAGCTGCGCCAGCGCTTGGCCGCGCGCCTTGAGGCTCTCAAGGAGAACGGCGGCGCCAGACTGGCCGAGTACCACGCCAAGGCCACCGAGCATCTGAGCACGCTCAGCGAGAAGGCCAAACCCGCGCTCGAGGACCTCCGCCAAGGCCTGCTGCCCGTGCTGGAGAGCTTCAAGGTCAGCTTCCTGAGCGCTCTCGAGGAGTACACTAAGAAGCTCAACACCCAGTAATAAGCTTGC

TABLE 12 MSP1D4-5 (helices 4 and 5 deleted) amino acid sequence (SEQ IDNO:23). MGHHHHHHIEGRLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESF KVSFLSALEEYTKKLNTQ

Deletion of helices 5 and 6 was performed in a similar manner, but twoseparate PCR steps using the following primers were employed in a firstreaction (Reaction 1, Primer C: 5-CAGAATTCGCTAGCCGAGTACCAC GCCAA-3′, SEQID NO:24; and Primer D: 5′-GCAAGCTTA TTACTGGGTGTTGAGCTTCTT-3′, SEQ IDNO:25) and a second reaction (Reaction 2, Primer E:5′-ATACCATGGGCCATCATCATC ATCATCATA-3′, SEQ ID NO:26; and Primer F:5′-CAGAATTC GCTAGCCTGGCGCTCAACTTCTCTT-3′, SEQ ID NO:27.

The PCR products encode the NB and C-terminal portions of MSP bothlacking helices 5 and 6 and each contain a NheI restriction site. Afterdigestion of the PCR products with Nhe I, NcoI and HindIII, thefragments was ligated into NcoI+HindIII treated pET 28 to produce theDNA sequence of MSP1 D5-6 lacking helices 5 and 6. See FIGS. 9A-9B.TABLE 13 MSP1D5-6 (helices 5 and 6 deleted) DNA sequence (SEQ ID NO:28).Translation start and stop codons are shown in bold type, andrestriction endonuclease recognition sites used in cloning areunderlined. TATACCATGGGCCATCATCATCATCATCATATAGAAGGAAGACTAAAGCTCCTTGACAACTGGGACAGCGTGACCTCCACCTTCAGCAAGCTGCGCGAACAGCTCGGCCCTGTGACCCAGGAGTTCTGGGATAACCTGGAAAAGGAGACAGAGGGCCTGAGGCAGGAGATGAGCAAGGATCTGGAGGAGGTGAAGGCCAAGGTGCAGCCCTACCTGGACGACTTCCAGAAGAAGTGGCAGGAGGAGATGGAGCTCTACCGCCAGAAGGTGGAGCCGCTGCGCGCAGAGCTCCAAGAGGGCGCGCGCCAGAAGCTGCACGAGCTGCAAGAGAAGTTGAGCGCCAGGCTAGCCGAGTACCACGCCAAGGCCACCGAGCATCTGAGCACGCTCAGCGAGAAGGCCAAACCCGCGCTCGAGGACCTCCGCCAAGGCCTGCTGCCCGTGCTGGAGAGCTTCAAGGTCAGCTTCCTGAGCGCTCTCGAGGAGTACACTAAGAAGCTCAACACCCAGTAATAAGCTTGC

TABLE 14 MSP1D5-6 (helices 5 and 6 deleted) amino acid sequence (SEQ IDNO:29). MGHHHHHHIEGRLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESF KVSFLSALEEYTKKLNTQ

Example 2 Construction of Synthetic MSP Gene.

A synthetic gene for MSP1 is made using the following overlappingsynthetic oligonucleotides which are filled in using PCR. The codonusage has been optimized for expression in E. coli, and restrictionsites have been introduced for further genetic manipulations of thegene. Synthetic nucleotide taps1a (SEQ ID NO:30)TACCATGGGTCATCATCATCATCATCACATTGAGGGACGTCTGAAGCTGTTGGACAATTGGGACTCTGTTACGTCTA Synthetic nucleotide taps2a (SEQ ID NO:31)AGGAATTCTGGGAGAACCTGGAAAAAGAAACCGAGGGACTGCGTCAGGAA ATGTCCAAAGATSynthetic nucleotide taps3a (SEQ ID NO:32)TATCTAGATGACTTTCAGAAAAAATGGCAGGAAGAGATGGAATTATATCG TCAA Syntheticnucleotide taps4a (SEQ ID NO:33)ATGAGCTCCAAGAGAAGCTCAGCCCATTAGGCGAAGAAATGCGCGATCGCGCCCGTGCACATGTTGATGCACT Synthetic nucleotide taps5a (SEQ ID NO:34)GTCTCGAGGCGCTGAAAGAAAACGGGGGTGCCCGCTTGGCTGAGTACCAC GCGAAAGCGACAGAASynthetic nucleotide taps6a (SEQ ID NO:35)GAAGATCTACGCCAGGGCTTATTGGCTGTTCTTGAGAGCTTTAAAGTCAG TTTTCT Syntheticnucleotide taps1b (SEQ ID NO:36)CAGAATTCCTGCGTCACGGGGCCCAGTTGTTCGCGAAGTTTACTGAAGGT AGACGTAACAG Syntheticnucleotide taps2b (SEQ ID NO:37)TCATCTAGATATGGCTGAACCTTGGCCTTCACCTCTTCTAAATCTTTGGA CATTT Syntheticnucleotide taps3b (SEQ ID NO:38)TGGAGCTCATGGAGTTTTTGGCGTGCCCCCTCTTGCAGTTCCGCACGCAGCGGTTCCACCTTTTGACGATATAATTCCAT Synthetic nucleotide taps4b (SEQ IDNO:39) GCCTCGAGACGTGCGGCCAAACGCTGGCGAAGTTCATCCGAATACGGCGCCAAATGAGTCCGGAGTGCATCAACAT Synthetic nucleotide taps5b (SEQ ID NO:40)GTAGATCTTCCAGCGCCGGTTTCGCTTTTTCGCTCAAGGTGCTCAGGTGT TCTGTCGCTTT Syntheticnucleotide taps6b (SEQ ID NO:41)CCAAGCTTATTACTGGGTATTCAGCTTTTTAGTATATTCTTCCAGAGCTG ACAGAAAACTGACTTT

TABLE 15 Full synthetic gene sequence for MSP1 (SEQ ID NO:42).Restriction sites used in cloning are underlined, and the translationstart and stop signals are shown in bold.ACCATGGGTCATCATCATCATCATCACATTGAGGGACGTGTGAAGCTGTTGGACAATTGGGACTCTGTTACGTCTACCTTCAGTAAACTTCGCGAACAACTGGGCCCCGTGACGCAGGAATTCTGGGACAACCTGGAAAAAGAAACCGAGGGACTGCGTCAGGAAATGTCCAAAGATTTAGAAGAGGTGAAGGCCAAGGTTCAGCCATATCTAGATGACTTTCAGAAAAAATGGCAGGAAGAGATGGAATTATATCGTCAAAAGGTGGAACCGCTGCGTGCGGAACTGCAAGAGGGGGCACGCCAAAAACTCCATGAGCTCCAAGAGAAGCTCAGCCCATTAGGCGAAGAAATGCGCGATCGCGCCCGTGCACATGTTGATGCACTCCGGACTCATTTGGCGCCGTATTCGGATGAACTTCGCCAGCGTTTGGCCGCACGTCTCGAGGCGCTGAAAGAAAACGGGGGTGCCCGCTTGGCTGAGTACCACGCGAAAGCGACAGAACACCTGAGCACCTTGAGCGAAAAAGCGAAACCGGCGCTGGAAGATCTACGCCAGGGCTTATTGCCTGTTCTTGAGAGCTTTAAAGTCAGTTTTCTGTCAGCTCTGGAAGAATATACTAAAAAGCTGAATACCCAGTAATAAGCTTG G

The following is the amino acid sequence of a MSP polypeptide in whichhalf repeats are deleted: TABLE 16 MSP with first half-repeat deleted(MSP1delta1) (SEQ ID NO:43)MGHHHHHHIEGRLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSILSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNT Q

TABLE 17 MSP with second half-repeat deleted (MSP1delta2) (SEQ ID NO:44)MGHHHHHHIEGRLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPVLESFKVSFLSALEEYTKKLNT Q

TABLE 18 MSP tandem repeat with first half-repeats deleted (MSP2delta1)(SEQ ID NO:45) MGHHHHHHIEGRLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQGTLKLLDNWDSVTSTFSKLREQLGPVTQEFWDNLEKETEGLRQEMSPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ

Other constructs that can be readily produced include permutations ofthe above, i.e. MSP1 or MSP2 with any combination of the following:hinge deletion, hinge replacement, half-repeat deletion, histidine tag,different linkers for MSP2.

Example 3 Expression of Recombinant MSPs

To express MSP proteins, the nucleic acid constructs were insertedbetween the NcoI and HindIII sites in the pET28 expression vector andtransformed into E. coli BL21(DE3). Transformants were grown on LBplates using kanamycin for selection. Colonies were used to inoculate 5ml starter cultures grown in LB broth containing 30 μg/ml kanamycin. Foroverexpression, cultures were inoculated by adding 1 volume overnightculture to 100 volumes LB broth containing 30 μg/ml kanamycin and grownin shaker flasks at 37° C. When the optical density at 600 nm reached0.6-0.8, isopropyl b-D-thiogalactopyranoside (IPTG) was added to aconcentration of 1 mM to induce expression and cells were grown 3-4hours longer before harvesting by centrifugation. Cell pellets wereflash frozen and stored at −80° C.

Example 4 Purification of Recombinant MSPs

Purification of histidine-tagged MSPs was carried out as follows. Afrozen cell pellet from 1 liter of expression culture was resuspended in25 milliliters of 20 mM Tris HCl pH 7.5 containing 1 mMphenylmethylsulfonyl fluoride. Triton X-100(t-octylphenoxypolyethoxyethanol) was added from a 10% (w/v) stock indistilled H20 to a final concentration of 1%. The resuspended cells weresonicated on ice at 50% duty cycle at a power setting of 5 for fourcycles of 1 minute on, 5 minutes off with a Branson probe sonifier.

The resulting lysate was centrifuged for 30 minutes at 30,000 rpm in aBeckman Ti 45 rotor in a ultracentrifuge. The resulting supernatant wasfiltered through a 0.22 mm nylon syringe filter. The salt concentrationwas adjusted to 0.5 M from a 4 M NaCl stock in water and applied to a 5ml Hi-Trap nickel loaded column (Pharmacia, Piscataway, N.J.).

For 6H-MSP1, the column is washed with 20 ml buffer (10 mM Tris pH 8,0.5 M NaCl) containing 1% Triton X-100, followed by 20 ml buffer+50 mMsodium cholate, and then 20 ml buffer and 20 ml 100 mM imidazole inbuffer. The His-tagged polypeptide is eluted with 15 ml 0.5 M imidazolein buffer.

For 6H-MSP2, the column is washed with 20 ml buffer (10 mM Tris pH 8,0.5 M NaCl) containing 1% Triton X-100; 20 ml buffer +50 mM cholate; 20ml buffer; 20 ml 35 mM imidazole in buffer. The His-tagged polypeptideis then eluted with 15 ml 0.5 M imidazole in buffer, and the purifiedprotein is dialyzed against 10 mM Tris pH 8, 0.15 M NaCl using a 10,000MW cutoff cellulose dialysis membrane.

Example 6 Production of MSP-Containing Nanoscale Particles

To reconstitute MSP proteins of the present invention with lipid,purified MSP was concentrated in a pressurized ultrafiltration device(Amicon) using a 10,000 MW cutoff filter to ˜2-6 mg protein/ml.Concentration of protein was determined by bicinchoninic acid assay(Pierce Chemical, Rockford, Ill.) or measurement of A280 usingtheoretical absorption coefficient. Phospholipid (dipalmitoylphosphatidylcholine in this case, however different phosphatidylcholinesand mixtures of phosphatidylcholine and other lipids can be used) inchloroform stock solution was dried under a stream of nitrogen andplaced in vacuo overnight. Phosphate analysis was performed to determinethe concentration of chloroform stock solutions. The dried lipid filmwas resuspended in buffer 10 mM Tris HCl pH 8.0 or pH 7.5 containing0.15 M NaCl and 50 mM sodium cholate to give a final lipid concentrationof 25 mM. The suspension was vortexed and heated to 50° C to obtain aclear solution. Phospholipid solution was added to solution of MSP (2-6mg/ml protein) to give molar ratios for MSP1:lipid of 2:200 and for MSP2of 1:200. The mixture was incubated overnight at 37° C. and thendialyzed against 1000 volumes of buffer without cholate with 4 changesof buffer over 2-3 days.

Example 7 Tethered Membrane Protein Incorporation

Tissue Factor (TF) is a representative tethered membrane protein. Inorder to demonstrate the value of MSP technology for a tethered membraneprotein, recombinant human TF was incorporated into MSP-supportednanodiscs. The recombinant protein consists of an extracellular domain,the transmembrane anchor and a truncated cytosolic domain. Thetruncation increases the homogeneity of the protein by removing theC-terminal portions of the protein which are subject to proteolysis bybacterial enzymes. This modification does not affect TF activity.Additional modifications to the protein include an N-terminaltrafficking peptide and an HPC4 epitope tag. The trafficking peptidedirects the expressed protein to the intermembrane space of therecombinant E. coli host cell, in which space the peptide sequence iscleaved. The HPC4 epitope allows for affinity purification with Ca²⁺dependent antibody (Rezaie et al., 1992) and does not affect TFactivity.

A 25 mM lipid mixture containing 80% phosphatidyl choline and 20%phosphatidyl serine was solubilized with 50 mM cholate in 10 mM Tris Cl,150 mM NaCl at pH 8.0. TF, MSP1 and lipid (in a ratio of 1:10:1000) werecombined and incubated overnight at 37° C. The sample was then dialyzedat 37° C. (10,000 dalton molecular weight cutoff membrane) againstbuffer containing 10 mM Tris Cl, 150 mM NaCl at pH 8.0 (lacking cholate)for 2 hours. Dialysis was then continued at 4° C. for an additional 6hours with buffer changes every 2 hours. The approximately 1 ml samplewas then concentrated to <250 μl using a YM-10 centrifuge concentratorand injected into a Pharmacia 10/30 Superdex 200 HR gel filtrationcolumn. Samples were eluted with buffer identical to that describedabove (no cholate) at 0.5 ml per minute. Fractions from chromatographywere run on an 8-25% gradient SDS polyacrylamide gel to determineapparent size and then checked for coagulation activity. Thechromatogram showing elution of TF incorporated into an excesspopulation of MSP1 nanodiscs is shown in FIG. 16A-16B.

The activity of TF in several disk fractions was determined bycoagulation assays with human serum. Activity was monitored in fractions25-28 as the inverse of coagulation time. Activity was highest infraction 25 at 40 hr⁻¹ and decreased through fraction 28 at 30 hr⁻¹.This is expected from the size chromatogram in that the leading edge ofthe nanodisc peak has a larger effective mass due to the incorporationof TF in the MSP-supported bilayer. This assay thus demonstrates that TFis incorporated into nanodiscs in an active conformation and that themembrane environment of the nanodisc closely mimics that of the nativemembrane system.

Cytochrome b5 is a membrane anchored heme protein having a singlemembrane anchor domain that penetrates the membrane bilayer. Cytochromeb5 solubilized from its native membrane exists as large aggregates inthe absence of detergent and appears as a smear rather than a discreteband on native polyacrylamide gel electrophoresis. Formation ofnanodiscs through a self-assembly process wherein cytochrome b5 is addedto the preparation of MSP and phospholipid results in the incorporationof cytochrome b5 into nanodisc structures. This is verified by theintense heme staining of the band corresponding to nanodiscs in theright panel of See FIG. 17B, lane 4. Cytochrome b5-containing nanodiscsseparated by anion exchange chromatography are shown in lanes 5 and 6 ofFIG. 17B. Two peaks elute from the anion exchange column near 310 mMNaCl and near 370 mM NaCl. Discs alone elute near 310 mM NaCl, andcytochrome b5 alone elutes between 450 and 700 mM NaCl. These data showthat cytochrome b5 can be successfully solubilized using MSP technologyand that disc complexes containing cytochrome b5 can bechromatographically separated and purified away from the undesiredaggregated material. The optical absorption properties of the hemechromophore of the purified material demonstrate that the heme activesite in a native conformation.

Nanodiscs can also be formed by mixing 20 μl of apo A-I (10 mg/ml), 6.6μl cytochrome b5 (0.5 mM) and 50 μl egg phosphatidylcholine/sodiumcholate (11.2 egg PC, 6.2 mg/ml sodium cholate), incubating overnight at4° C., followed by dialyzing to remove cholate. Purification wasaccomplished using a Pharmacia MonoQ FPLC anion exchange columnequilibrated in 25 mM Tris Cl, pH 8.0. A linear gradient was run at 0.5ml/min from 0-1 M NaCl in 20 min.

Example 8 Embedded Membrane Protein Incorporation

Cytochrome P450 2B4 from rabbit liver microsomes, cytochrome P450 3A4found in nature in human liver microsomes and cytochrome P450 6B1 frominsect microsomes are representative of embedded membrane proteins.

Cytochrome P450 2B4 was isolated from rabbit liver microsomes afterinduction with phenobarbital. Formation of 2B4 nanodiscs is as follows.Cytochrome P450 2B4 was reconstituted into disks by the detergentdialysis method. The buffer consisted of 10 mM Tris-HCl pH 8.0, 0.1 MNaCl, 10% (v/v) glycerol. The mixture of apo A-I, cholate andphospholipid (1:220:110 mole ratio) was incubated for 8 hours at 37° C.followed by addition of P450 (1:0.5, apo A-I:P450 mole ratio) andincubation overnight at room temperature. The mixture was dialyzed usinga 10,000 MW cutoff slide-a-lyzer (Pierce Chemical Co., Rockford, Ill.)at room temperature for two hours followed by a change of buffer andcontinued dialysis at 4° C. It was found that 82% of the P450 contentcould be recovered under these conditions. After dialysis, the mixturewas injected onto a Superdex 200 HR10/30 gel filtration column(Pharmacia, Uppsala, Sweden) equilibrated in reconstitution buffer atroom temperature at a flow rate of 0.25 ml/minute with collection of 0.5ml fractions. Fractions were assayed using native polyacrylamidegradient gel electrophoresis on 8-25% gradient native gels and Coomassiestaining using the Phastgel system (Pharmacia, Uppsala, Sweden).

Human cytochrome P450 3A4, normally from liver microsomes, has also beencloned, expressed in E. coli and purified and incorporated intoMSP-supported bilayer nanodiscs. 10 nanomoles of MSP2, one micromole oflipid, 5 nanomoles of cytochrome P450 3A4 protein and 2 micromolescholic acid were incubated together at 37° C. for 2 hours. The incubatedmixture was then dialyzed in a 10K Slide-A-lyzer Dialysis Cassette(Pierce Chemical Co., Rockford, Ill). The dialysis was carried out with10 mM potassium phosphate (pH 7.4) 150 mM NaCl buffer. The sample wasdialyzed at 37° C. for 6 hours followed by a buffer change, and dialysiscontinued at 4° C. with two buffer changes at 12 hour intervals. Thesamples were then fractionated on a Superdex 200 HR 10/30 column(Pharmacia, Uppsala, SE) equilibrated in dialysis buffer at roomtemperature at a flow rate of 0.5 ml/min.

The four graphs (FIGS. 18-20) show that the retention times of thecytochrome P450 3A4 (observed by absorbance at 417 nm) and the nanodiscs(monitored at 280 nm where both MSP and the 3A4 protein absorb) elutefrom the column at the same time, at approximately 24 min. This elutiontime also correlates closely to the calculated retention time of thedisc protein complex. Further evidence that supports this is a nativepolyacrylamide gel electropherogram that directly measures the size ofthe eluted particles (FIG. 21).

Cytochrome P450 6B1 is another model embedded membrane protein. Thiscytochrome has been isolated from Papilio polyxenes, the blackswallowtail. These butterflies feed exclusively on plants producingfuranocoumarins, plant metabolites that are phototoxic to mostorganisms. Cytochrome 6B1 catalyzes the detoxification offuranocoumarins.

In order to show a new utility of the MSP methodology of the presentinvention, we demonstrated that isolated membranes containing theirrepertoire of membrane proteins could be sued as a source forincorporating membrane proteins into nanodiscs. An importantillustrative embodiment is the use of the common insect cell(Sf9)-baculovirus expression system which is used widely as aheterologous expression system. Thus, we used an insect cell lineco-infected such that a microsomal preparation containing overexpressedinsect 6B1 and also overexpressed insect NADPH cytochrome P450reductase. In these experiments we not only demonstrate that MSPnanodiscs can be used to incorporate another cytochrome P450 system intosoluble monodisperse particles but also that the source of this P450could be simply whole membranes containing this protein.

A standard baculovirus expression system was used to obtain microsomalpreparations with overexpressed insect cytochrome 6B1 and insect NADPHP450 reductase. Based on the lipid concentration contained in themicrosomal preparations, MSP technology was used to assemble microsomalproteins into nanoparticle discs using a ratio of 110:1:220lipid:MSP1:cholate. The microsomal sample was detergent solubilized withcholate and mixed with MSP1. The sample was incubated at 4° C. for 2hours. The detergent can be removed by dialysis or hydrophobic beads. Inthis experiment BIOBEADS (hydrophobic beads, BioRad, Hercules, Calif.)were added in excess (0.25 g per 1 ml disc mixture) and incubated for 2hours at 4° C. for 2 hours to remove detergent. The sample was removedfrom the beads and the His₆-tagged MSP was isolated by using a batchpurification method with Ni²⁺ resin. The MSP discs were then isolated bySuperdex sizing column chromatography (FIG. 22). Incorporation of P450into the His₆-tagged discs was followed by CO difference spectroscopy ofnickel affinity column purified and sizing column-purified fractions(FIG. 24). SDS-PAGE was performed to verify incorporation of cytochromeP450 6B1 into discs (FIG. 23).

The endogenous (natural) ratio of cytochrome P450 to reductase is about10-20. To obtain activity of the cytochrome P450 6B1 afterreconstitution into discs, it is preferred that an excess of reductasebe added to the reconstitution mixture, such that a P450 molecule andreductase molecule both partition into a single disc. Supplementation ofthe microsomal preparation with exogenously added reductase has beensuccessfully demonstrated.

The protocol for making discs using microsomal preparations was usedwith one modification. Exogenous rat reductase was added after thesolubilization step of the microsomal preparation with cholate andbefore the addition of MSP1. Otherwise identical disc assembly andpurification procedures were followed. The sample was separated by aSuperdex sizing column, where absorbance at 280 nm indicates thepresence of MSP1, absorbance at 420 and 456 nm indicates the presence offerric species, and absorbance at 456 nm also indicates presence ofreductase. A ratio plot of 456 to 420 nm was made; it showed positionson the chromatogram where the absorbance at 456 nm was above thatassociated with cytochrome P450 6B1 and, therefore, could be attributedto absorbance by reductase. Retention times reflected the presence of 10nm particles containing cytochrome P450 6B1 and reductase (FIG. 26).

MSP-supported nanodiscs with purified proteins, membrane fragments ordisrupted membranes can be used in high throughput screening ventures,for example, to identify new pharmaceuticals and other biologicallyactive molecules.

Example 9 Integral Membrane Protein Incorporation

Bacteriorhodopsin is a model integral membrane protein.Bacteriorhodopsin was incorporated into nanoscale structures using thefollowing procedure, which is a protocol useful for other proteins aswell. Bacteriorhodopsin was obtained as lyophilized purple membrane fromSigma (St. Louis, Mo.). 1 mg BR was suspended in 1 ml 25 mM potassiumphosphate pH 6.9. 1 ml 90 mM n-octyl B-D-glucopyranoside in the samebuffer was added and the sample placed in the dark at 24° C. overnight.This treatment produces a detergent-solubilized monomeric form (Dencheret al., 1982). BR was quantitated assuming a molar extinctioncoefficient at 550 nm of 63,000. BR (7.8 uM) was mixed with MSP1 (97 mM)or MSP2 (110 mM) and cholate (50 mM) to give final molar ratios ofMSP1:BR of 10:1 or MSP2:BR of 5:1 and a cholate concentration ofapproximately 8 mM. For reconstitution with phospholipid, the lipid issolubilized as above in the presence of 50 mM cholate and mixed withMSP1 at a mole ratio of 1 MSP1:110 lipids:0.1 BR. The mixture wasincubated at room temperature for ˜3 hours followed by dialysisovernight against 1000 volumes of buffer using 10,000 MW cutoff dialysisdevices (Slide-a-lyzer, Pierce Chemical). Dialysis was continued at 4degrees for 2 days with several changes of buffer. 10 mM HEPES, pH 7.5,0.15 M NaCl buffer can be used. Tris buffer pH 7.5 or pH 8 has also beenused successfully.

The 5-hydroxytryptamine 1A G protein coupled receptor from human hasbeen incorporated into MSP-containing nanoparticles. A commerciallyavailable insect cell expression system that provides a membranefraction containing the human 5-hydroxytryptamine 1A GPCR was supportedusing MSP compositions. Briefly, the 5-HT receptor containing membranepreparation was mixed with phospholipids (phosphatidyl choline,phosphatidylethanolamine, phosphatidyl serine) at a ratio of 45:45:10,MSP1 and cholate.

5-HT1A receptors overexpressed in a commercially available Sf9 insectcell membrane preparation (Sigma Chemical Co., St. Louis, Mo.) weresolubilized using the following protocol. POPC, POPS and POPE (AvantiPhospholipids) in chloroform were mixed in a 45:10:45 mole ratio anddried down under a stream of nitrogen, then placed under vacuum forseveral hours to remove residual solvent. The phospholipids weredispersed in 50 mM Tris pH 7.4, 0.2 M NaCl, 50 mM sodium cholate bufferat a concentration of 25 mM phospholipid. Five microliters of the sf9membrane preparation (0.2 mg/ml protein), 1.62 microliters ofphospholipid in buffer, 2.4 microliters of MSP1 (4.2 mg/ml) and 0.28microliters 4 M NaCl were mixed and left for 1 hour on ice. The mixturewas diluted to 100 microliters total volume with 50 mM Tris pH 7.4 anddialyzed in a mini slide-a-lyzer (Pierce Chemical) against 50 mM Tris pH7.4 at 4° C. (two one-liter changes of buffer). To determine the amountof 5HT1A receptor associated with nanodisks, a radiolabeled ligand wasbound to the receptor and disk-receptor-ligand complexes were isolatedusing the 6-histidine tag present in the MSP1 according to the followingprotocol. After dialysis, the mixture was diluted to 200 microliterstotal volume with 50 mM Tris pH 7.4. Ninety-five microliters of thediluted mixture were placed into each of two tubes. One hundred fivemicroliters of stock reagent were added to give final concentrations of50 mM Tris pH 7.4, 10 mM MgSO4, 0.5 mM EDTA, 0.1% ascorbic acid in afinal volume of 200 microliters. Tritium-labeled 8-hydroxy-DPAT(specific activity 135000 Ci/mole) was added to each tube to give aconcentration of 1.5 nM. As a control, unlabeled metergoline (finalconcentration 100 micromolar) was added to one of the tubes as acompetitive ligand. After 1 hour on ice, the mixture was applied to 200microliters of Ni-chelating resin to specifically bind receptorassociated with 6Histidine-tagged MSP1 disks. The resin was washed threetimes with 0.5 ml of cold 50 mM Tris pH 7.4 to remove non-specificallybound ligand. Specifically bound radiolabeled 8-hydroxy-DPAT bound toreceptor/disk complexes was eluted with 0.5 ml 0.5 Molar imidazole in 10mM Tris pH 7.4, 0.5 M NaCl. Scintillation cocktail was mixed with theeluate and specifically bound radioligand was determined byscintillation counting. Between five and fifteen percent of the receptorinitially present in the sf9 membrane was found to be associated withMSP1 nanodisks.

The particles into which the 5-HT GPCR had incorporated were dialyzed.Functionality (in terms of ligand binding) was tested using dialysisagainst buffer containing tritiated 8-OH-DPAT, an agonist of thisreceptor. The particles were then run over a Ni—NTA column to bind viathe histidine tag on the MSP1 and to separate the particles from8-OH-DPAT which had not bound to the particles, and the material boundto the column was then eluted. Association of the tritium labeledagonist was demonstrated, showing that the incorporated GPCR retainedits ability to bind agonist.

Example 10 Analysis of MSP-supported Nanodisc Phospholipid Assemblies

The particles resulting from self assembly of membrane scaffold proteinsand phospholipids, either with or without an additional target protein,were analyzed as follows.

Bacteriorhodopsin-containing particles were dialyzed, and the resultingmixture was injected onto a Superdex 200 HR10/30 gel filtration column(Pharmacia) and eluted with buffer at 0.5 ml/min at room temperature.Absorbance was monitored at 280 nm for protein and 550 nm for BR. 0.5 mlfractions were collected. The column was calibrated using a mixture ofthyroglobulin (669 kDa, Stoke's diameter 170 A), ferritin (440 kDa,Stoke's diameter 122 A), catalase (232 kDa, Stoke's diameter 92 A),lactate dehydrogenase (140 kDa, Stoke's diameter 82 A), bovine serumalbumin (66 kDa, Stoke's diameter 71 A), and horse heart cytochrome c(12.4 kDa, Stoke's diameter 35.6 A).

Atomic Force Microscopy (AFM) was performed with a Digital InstrumentsNanoscope IIIa in contact mode with sharpened silicon nitride probesunder buffer. MSP1 and MSP2 dipalmitoyl phosphatidylcholine particleswere treated with 1:50 Factor Xa:MSP protein by mass in 10 mM Tris pH 8,0.15 M NaCl, 2 mM CaCl₂ for 8 hours. 2-10 ml sample was placed on afreshly cleaved mica surface along with 20 ml imaging buffer (10 mM TrispH 8, 0.15 M NaCl, 10 mM MgCl₂) and incubated for 30 minutes or longerbefore mounting sample in the fluid cell. Several milliliters of bufferwere flushed through the fluid cell to remove unadsorbed material.

Phosphate analysis of the nanoscale particles was carried out asfollows. Phosphate assay procedures were adapted from Chen et al. (1956)Anal. Chem. 28:1756-1758 and Fiske and Subbarow (1925). Samplescontaining roughly 40 nmoles lipid phosphate were dried down in glasstubes. 75 ml 8.9 N H₂SO₄ was added to each tube and heated to 210° C.for 30 minutes. 1 drop 30% H₂O₂ was added to each tube and heated for 30minutes. Tubes were cooled, 0.65 ml H₂O was added followed by 83.3 ml2.5% w/v ammonium molybdate tetrahydrate followed by vortexing and theaddition of 83.3 ml 10% w/v ascorbic acid. After mixing, the tubes wereplaced in a boiling water bath for 7 minutes. Absorbance was read at 820nm. Absorbance was calibrated using potassium phosphate standards from 0to 100 nmol phosphate. Buffer blanks from column chromatography wereincluded for MSP proteins.

Example 12 MSP-Supported Structures on Surfaces

Nanodiscs comprising MSPs and a protein of interest can be assembledonto a gold surface. The utility of this relates to the resultingepitaxial presentation of a target incorporated into a nanodisc assemblyto the solution. This offers an ideal system for quantitating binding ofother macromolecules or small molecules tagged with dielectric contrastagents to the target protein. A common methods of accomplishing suchmeasurements uses surface plasmon resonance (SPR) technology. SPR is acommon technique used to monitor biomolecular interactions at surfaces.The ability of SPR to rapidly detect and quantitate unlabeled proteininteractions on gold surfaces is useful for creating high through putchip assays for diverse membrane proteins (embedded and solubilized) ondiscs.

Discs consisting of the phospholipid DPPC either with or without anadditional thiolated lipid and MSP1 protein were prepared as follows. A25 mM lipid mixture containing phosphatidylcholine was solubilized with50 mM cholate in 10 mM Tris Cl, 150 mM NaCl at pH 8.0 were combined andincubated overnight at 37° C. For thiolated discs, 90%phosphatidylcholine and 10% thiolated lipid (ATA-TEG-DSPA, NorthernLipids) was solubilized in 3.3 mM Tris Cl, 66.7 mM borate, 150 mM NaClat pH 9.0 in order to unmask the thiols in the thiolated lipids. MSP1and lipid (1:100) were combined and incubated overnight at 37° C. Thesample was then dialyzed at 37° C. (10,000 MW cutoff membrane) againstbuffer containing 10 mM Tris Cl, 150 mM NaCl at pH 8.0 without cholatefor 2 hours. Dialysis was then continued at 4° C. for an additional 6hours with buffer changes every 2 hours. The approximately 1 ml samplewas concentrated to <250 μl using a YM-10 centrifuge concentrator andinjected onto a Pharmacia 10/30 Superdex 200 HR gel filtration column.Samples were eluted from the column using the stated buffer withoutcholate at flow rates of 0.5 ml/min. Fractions from chromatography wereanalyzed by polyacrylamide gel electrophoresis using 8-25% gradientpolyacrylamide gel to determine apparent size.

The nanodisc samples (3-20 μM) prepared as described were injected intoan SPR instrument to determine if the discs would bind to the goldsurface. Both the DPPC and 10% thiolated lipid discs adsorbed to a goldsurface and a modified gold surface covered with a monolayer of methylterminated thiol (nonanethiol) or carboxyl terminated thiol(11-mercaptoundecanoic acid). Thiolated discs were injected using abuffer consisting of 3.3 mM Tris, 66.7 mM borate, 150 mM NaCl, pH 9.0.DPPC discs were injected using a buffer of 10 mM Tris, 150 mM NaCl, pH7.5 or pH 8.0. In all cases, the discs could not be removed even underharsh conditions (0.5 M HCl). Surface coverage was shown to increasewith increasing concentration of discs injected (3 μM vs. 19 μM). Discsdo not form perfectly packed monolayers; accordingly, surface coverageis limited by the jamming limit (theoretical maximum coverage based onrandom sequential absorption to the surface modeling discs as identicalnon-overlapping hard spheres) of 0.547. The coverage for a fullmonolayer of discs was calculated based on an assumption of disc heightof 5.5 nm and a refractive index between 1.45 and 1.5. The fullmonolayer values were multiplied by the jamming limit to determine themaximum coverage that was then used to determine percent coverage basedon experimental values. When the disc concentration was at least 10 μM,the estimated coverages were between about 62 and about 103%. Theresultant SPR trace demonstrating association of the nanodiscs to thegold surface is shown in FIG. 27.

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1. A nanoscale particle comprising an artificial membrane scaffoldprotein and at least one tethered membrane protein, and furthercomprising a phospholipid or a mixture of phospholipids, wherein saidnanoscale particle has a diameter between 5 nm and 500 nm, wherein saidartificial membrane scaffold protein, in an aqueous environment,self-assembles in the presence of a phospholipid or in the presence of amixture of phospholipids, into a nanoscale particle between about 5 nmand about 500 nm in diameter, wherein said membrane scaffold protein isamphipathic and wherein said membrane scaffold protein is a derivativeor a truncated form of human apolipoprotein A1, forms at least one alphahelix, and lacks the N-terminal globular domain of human apolipoproteinA1 (amino acids 8-50 of SEQ ID NO:2).
 2. The nanoscale particle of claim1, wherein said tethered membrane protein is a NADPH-cytochrome P450reductase.
 3. The nanoscale particle of claim 1, wherein said tetheredmembrane protein is a cytochrome b5.
 4. The nanoscale particle of claim1, wherein said artificial membrane scaffold protein comprises an aminoacid sequence selected from the group consisting of SEQ ID NO:6, SEQ IDNO:9, SEQ ID NO:17, amino acids 13 to 414 of SEQ ID NO:17, SEQ ID NO:19,amino acids 13 to 422 of SEQ ID NO:19, SEQ ID NO:23, amino acids 13 to168 of SEQ ID NO:23, SEQ ID NO:29, amino acids 13 to 169 of SEQ IDNO:29, SEQ ID NO:43, amino acids 13 to 201 of SEQ ID NO:43, SEQ IDNO:44, amino acids 13 to 201 of SEQ ID NO:44, SEQ ID NO:45, and aminoacids 13 to 392 of SEQ ID NO:45.
 5. The nanoscale particle of claim 2,wherein said artificial membrane scaffold protein comprises an aminoacid sequence selected from the group consisting of SEQ ID NO:6, SEQ IDNO:9, SEQ ID NO:17, amino acids 13 to 414 of SEQ ID NO:17, SEQ ID NO:19,amino acids 13 to 422 of SEQ ID NO:19, SEQ ID NO:23, amino acids 13 to168 of SEQ ID NO:23, SEQ ID NO:29, amino acids 13 to 169 of SEQ IDNO:29, SEQ ID NO:43, amino acids 13 to 201 of SEQ ID NO:43, SEQ IDNO:44, amino acids 13 to 201 of SEQ ID NO:44, SEQ ID NO:45, and aminoacids 13 to 392 of SEQ ID NO:45.
 6. The nanoscale particle of claim 3,wherein said artificial membrane scaffold protein comprises an aminoacid sequence selected from the group consisting of SEQ ID NO:6, SEQ IDNO:9, SEQ ID NO:17, amino acids 13 to414 of SEQ ID NO:17, SEQ ID NO:19,amino acids 13 to 422 of SEQ ID NO:19, SEQ ID NO:23, amino acids 13 to168 of SEQ ID NO:23, SEQ ID NO:29, amino acids 13 to 169 of SEQ IDNO:29, SEQ ID NO:43, amino acids 13 to 201 of SEQ ID NO:43, SEQ IDNO:44, amino acids 13 to 201 of SEQ ID NO:44, SEQ ID NO:45, and aminoacids 13 to 392 of SEQ ID NO:45.